AU640619B2 - Hiv proteins and peptides useful in the diagnosis, prophylaxis or therapy of aids - Google Patents

Description

OPI DATE 01/05/90 APPLN. ID 44036 89 P I AOJP DATE 07/06/90 PCT NUMBER PCT/US89/04302 INTERNATIONAL APPLICATION PUBLISHED UNDER THE PATENT COOPERATION TREATY (PCT) (51) International Patent Classification 5 (11) International Publication Number: WO 90/03984 C07K 7/10, A61K 39/21 C12N 15/48, C12P 21/02, 21/08 Al (43) International Publication Date: 19 April 1990 (19.04.90) G01N 33/569 (21) International Application Number: PCT/US89/04302 Department of Biotechnology, 25, rue du Docteur-Roux, F- 75724 Paris Cedex 15 O'KEEFFE, Thomas 14 Karen (22) International Filing Date: 29 September 1989 (29.09.89) Road, Windham, NH 03089 LAROSA, Gregory Strathmore Road, Apt. 33, Brookline, MA 02146 PRO- Priority data: FY, Albert, T. 28 Essex Street, Cambridge, MA 02139 (US).

252,949 3 October 1988 (03.10.88) US 359,543 1 June 1989 (01.06.89) US (74)Agents: SALIWANCHIK, Roman et al.; Saliwanchik 407,663 19 September 1989 (19.09.89) US Saliwanchik, 2421 N.W. 41st Street, Gainesville, FL 32606 (US).

(71) Applicant: REPLIGEN CORPORATION [US/US]; One Kendall Square, Building 700, Cambridge, MA 02139 (81) Designated States: AT (European patent), AU, BE (European patent), CH (European patent), DE (European patent), DK, FI, FR (European patent), GB (European pa- (72) Inventors: RUSCHE, James, R. 18 Brigham Road, Fram- tent), IT (European patent), JP, KR, LU (European paingham, MA 01701 PUTNEY, Scott, D. 5 Epping tent), NL (European patent), NO, SE (European patent).

Street, Arlington, MA 02174 JAVAHERIAN, Kashayar 27 Webster Road, Lexington, MA 02173 Published FARLEY, John 507 Reeves Road, Pittsford, NY Wihntertion arcl ort.4 14534 GRIMAILA, Raymond 311 Washington Street, Somerville, MA 02134 LYNN, Debra 1 Watermill Place, Unit 328, Arlington, MA 02174 (US).

PETRO-BREYER, Joan Institut Pasteur, (54) Title: HIV PROTEINS AND PEPTIDES USEFUL IN THE DIAGNOSIS, PROPHYLAXIS OR THERAPY OF AIDS (57) Abstract The subject invention concerns the identification of a portion'of the HIV envelope protein called the principal neutralizing domain. Polypeptides comprising this domain have the capability of raising, and/or binding with, neutralizing antibodies. The invention further concerns novel HIV polypeptides which can be used in the diagnosis, prophylaxis, or therapy of AIDS. These polypeptides can be prepared by known chemical synthetic procedures, or by recombinant DNA means. The polypeptides pertain to the gp 120 subunit, amino acids 298-320, including the sequence gly-pro-gly...... and variants thereof. Multiepitope polypeptides comprising analogues of this peptide epitope from different HIV variants are referred to.

WO 90/03984 PCT1/US89/04302 1

DESCRIPTION

HTV PROTEINS AND PEPTIDES USEFUL IN THE DIAGNOSIS. PROPHYLAXIS OR THERAPY OF AIDS Cross-Reference to a Related Application This is a continuation-in-part of our co-pending application Serial No. 359,543, filed on June 1, 1989, which is a continuation-in-part of our co-pending application Serial No. 252,949, filed on October 3, 1988, which is a continuation-in-part of our co-pending application Serial No. 090,080, filed on August 27, 1987.

Background of the Invention Human immunodeficiency virus (HIV), human T-cell lymphotropic virus 111 (HTLV-III), lymphadenopathy-associated virus (LAV), or AIDS-associated retrovirus (ARV) has been identified as the cause of acquired immune deficiency syndrome (AIDS) (Popovic, M.G. Sarngadharan, E.

Read, and R.C. Gallo [1984] Science 224:497-500). The virus displays tropism for the OKT4+ lymphocyte subset (Klatzmann, F. Barre-Sinoussi, M.T. Nugeyre, C. Dauget, E. Vilmer, C. Griscelli, F. Brun-Vezinet, C. Rouzioux, J.D. Gluckman, J.C. Chermann, and L Montagnier [1984] Science 225:59-63). Antibodies against HIV proteins in the sera of most AIDS and AIDS related complex (ARC) patients, and in asymptomatic people infected with the virus (Sarngadharan, M. Popovic, L. Bruch, J. Schupbach, and R.C. Gallo [19841 Science 224:506-508) have made possible the development of immunologically based tests that detect antibodies to these antigens. These tests are used to limit the spread of HIV through blood transfusion by identifying blood samples of people infected with the virus. Diagnostic tests currently available commercially use the proteins of inactivated virus and antigens.

In addition to allowing new approaches for diagnosis, recombinant DNA holds great promise for the development of vaccines against both bacteria and viruses (Wilson, T. 11984] Bio/Technology 2:29-39). The most widely employed organisms to express recombinant vaccines have been E. coli, S cerevisiae and cultured mammalian cells. For example, subunit vaccines against foot and mouth disease (Kleid, D. Yansura, B. Small, D. Dowbenko, D.M. Moore, M.J. Brubmat., P.D.

McKercher, D.O. Morgan, B.H. Robertson, and H.L. Bachrach [19811 Science 214:1125-1129) and malaria (Young, W.T. Hockmeyer, M. Gross, W. Ripley-Ballou, R.A. Wirtz, J.H. Trosper, R.L.

Beaudoin, M.R. Hollingdale, LM. Miller, C.L Diggs, and M. Rosenberg 11985] Science 228:958-962) have been synthesized in E. coll. Other examples are hepatitis B surface antigen produced in yeast (McAleer, WJ., E.B. Buynak, R.Z. Maigetter, D.E. Wampler, WJ. Miller, and M.R. Hilleman 119841 Nature 307:178-180) and a herpes vaccine produced in mammalian cells (Berman, T. Gregory, D. Chase, and LA. Lasky 11994] Science 227:1490-1492).

WO 90/03984 PC/US9/04302 2 The entire HIV envelope or portions thereof have been used to immunize animals. The terms "protein," "peptide," and "polypeptide' have been used interchangeably in this application to refer to chemical compounds having more than one amino acid. The term "compound" as used here refers to chemical compounds in general. Thus, 'compound" includes proteins, peptides, and polypeptides. Also included under the category of "compound" are fusion compounds where polypeptides are combined with non-polypeptide moieties. As used in the present application, the term "naturally occurring HIV envelope protein' refers to the proteins gpl60, gpl20, and gp41 only. .As used in the present application, HIV refers to any HIV virus, including HIV-1 and HIV-2.

Both the native gpl20 (Robey et al. [1986] Proc. Natl. Acad. Sci. 83:7023-7027; Matthews et al. [1986] Proc. Natl. Acad. Sci. 83:9709-9713) and recombinant proteins (Laskey et al. [1986] Science 233:209-212; Putney et al. [1986] Science 234:1392-1395) elicit antibodies that can neutralize HIV in cell culture. However, all of these immunogens elicit antibodies that neutralize only the viral variant from which the subunit was derived. Therefore, a novel vaccine capable of protecting against multiple viral variants would be advantageous and unique.

HIV is known to undergo amino acid sequence variation, particularly in the envelope gene (Starcich, B.R. [1986] Cell 45:637-648; Hahn, B.H. et al. [1986] Science 232:1548-1553). Over 100 variants have been analyzed by molecular cloning and restriction enzyme recognition analysis, and several of these have been analyzed by nucleotide sequencing. Some of these are the HIV isolates known as RF (Popovic, M. et al. [1984] Science 224:497-500), WMJ-1 (Hahn, B.H. et- al. 11986] Science 232:1548-1553), LAV (Wain-Hobson, S. et al. [1985] Cell 40:9-17), and ARV-2 (Sanchez- Pescador, R. et al. [1985] Science 227:484-492). One aspect of this invention is defining the portion of HIV that comprises the principal neutralizing domain. The principal cntwralizing domain is located between the cysteine residues at amino acids 296 and 331 of the I .V savelope. The numbering of amino acids follows the published scquence of HIV-IIIg (Ratkte, L et al. [1985] Nature 313:277- 284). This domain is known to be hypervariable but retains the type-specific antigenic and immunogenic properties related to virus neutralization.

A further aspect of the subject invention is the discovery of highly conserved amino acids within the principal neutralizing domain. Although certain sequences from this region have been published (see, for example, Southwest Foundation for Biological Research, published PCT application, Publication No. WO 87/02775; Genetic Systems Corporation, Published United Kingdom Application No. GB 2196634 A; Stichting Centraal Diergeneeskundig lnstituut, Published EPO Application No.

0 311 219), the presence of the conserved regions described here have never before been described.

Diagnostic kits or therapeutic agents using viral proteins isolated from virus infected cells or recombinant proteins would contain epitopes specific to the viral variant from which they wer: isolated. Reagents containing proteins from multiple variants would have the utility of being .more broadly reactive due to containing a greater diversity of epitopes. This would be advantageous in the screening of serum from patients or therapeutic treatment of patients.

WO 90/03984 PCr/US89/04302 3 Synthetic peptides can be advantageous as the active ingredient in a vaccine, therapeutic agent or diagnostic reagent due to the ease of manufacture and ability to modify their structure and mode of presentation.

Synthetic peptides have been used successfully in vaccination against foot and mouth disease virus (Bittle et al. 11982] Nature 298:30-33); poliovirus (Emini et al. [1983] Nature 305:699); hepatitis B (Gerin et al. [1983] Proc. Natl. Acad. Sci. 80:2365-2369); and influenza (Shapira et al. [1984] Proc.

Natl. Acad. Sci. 81:2461-2465).

There is a real need at this time to develop a vaccine for AIDS. Such a vaccine, advantageously, would be effective to immunize a host against the variant AIDS viruses.

Brief Summary of the Invention The subject invention defines the location of the HIV principal neutralizing domain and discloses methods to utilize this segment of the HIV envelope protein for developing diagnostic, therapeutic, and prophylactic reagents. More specifically, the HIV principal neutralizing domain is located between cysteine residues 296 and 331 of the HIV envelope protein. The location of this domain is shown in Table 1. Although the specific amino acid sequence of the principal neutralizing domain is known to be highly variable between variants, we have found that peptides from this domain are invariably capable of raising, and/or binding with, neutralizing antibodies. This unexpected discovery provides a basis for designing compositions and strategies for the prevention, diagnosis, and treatment of AIDS.

The discovery of the principal neutralizing domain (also known as the "loop") resulted from extensive research involving a multitude of HIV envelope proteins and peptides from many HIV variants. Proteins and peptides capable of raising, and/or binding with, neutralizing antibodies are disclosed here. These novel HIV proteins and peptides, or their equivalents, can be used in the diagnosis, prophylaxis, and/or therapy of AIDS. Further, the peptides can be used as immunogens or screening reagents to generate or identify polyclonal and monoclonal antibodies that would be useful in prophylaxis, diagnosis and therapy of HIV infection.

A further aspect of the invention is the discovery of highly conserved regions within the principal neutralizing domain. This discovery was quite unexpected because of the known variability of the amino acids within this segment of the HIV envelope protein.

The proteins and peptides of the invention are identified herein by both their amino acid sequences and the DNA encoding them. Thus, they can be prepared by known chemical synthetic procedures, or by recombinant DNA means.

These peptides, or peptide, having the antigenic or immunogenic properties of these peptides, can be used, advantageously, in a vaccine, a cocktail of peptides, to elicit broad neutralizing antibodies in the host. Further, these peptides can be used sequentially, immunizing initially with a peptide equivalent to the principal neutralizing domain of an HIV variety followed by immunization WO 90/03984 PCT/US89/04302 4 with one or more of the above peptides. Polyclonal or monoclonal antibodies that bind to these peptides would be advantageous in prophylaxis or therapy against HIV, the causative agent of AIDS.

Brief Description of the Drawings Figure 1 shows commonly occurring sequences of the principal neutralizing domain.

Figure 2 is a schematic for multi-epitope gene construction.

Figure 3 depicts the steps in the construction of a specific multi-epitope gene.

Figure 4 shows the sequences of four synthesized single-stranded oligomers for construction of a multi-epitope gene.

Detailed Disclosure of the Invention Described here is a segment of the HIV envelope protein which raises, and/or binds with, neutralizing antibodies. This unique and highly unexpected property has been observed in each HIV variant that has been examined. The segment of interest has been named the "principal neutralizing domain." The principal neutralizing domain is bounded by cysteine residues which occur at positions 296 and 331. It should be noted that these same cysteine residues have been described as beginning at 302, rather than 296 (Rusche, J.R. et al. [1988] Proc. Natl. Acad. Sci. USA 85(15):3198-3202).

Because the cysteine residues are linked through disulfide bonds, the segment between the residues tends to form a loop. Therefore, the principal neutralizing domain is also referred to as the "loop." The segment of the protein envelope identified here as the principal neutralizing domain is known to be highly variable between HIV variants. Thus it is surprising that, for each variant, this segment is capable of eliciting, and/or binding with, neutralizing antibodies.

The principal neutralizing domain identified here is a small segment of the HIV envelope protein. This small segment may be combined with additional amino acids, if desired, for a specific purpose, All such proteins are claimed here except where such proteins constitute a naturally occurring HIV envelope protein. As used here, the term "naturally occurring envelope protein" refers only to gpl60, gpl20, and gp41.

Listed in Table 1 are sequences of the principal neutralizing domain for some of the variants tested. Table 9 contains a complete list of the principal neutralizing domains.

Amino acids may be referred to using either a three-letter or one-letter abbreviation system.

The following is a lis' of the common amino acids and their abbreviations: WO 90/03984PC/U8/42 PCr/US89/04302 Amno acid Alanine Arginine Asparagine Aspartic acid Asn and/or Asp Cysteine Glutamine Glutamic acid GIn and/or Glu.

Glycine Histidine Isoleucine Leucine Lysine Methionine Phenylalanine Proline Serine Threonine Tryptophan 'Tyrosine Valine Three-letter symbol Ala Arg Asn Asp Asx Cys Gin Glu

GIX

Gly His le Leu

LYS

Met Plie Pro Ser Thr Trp Tyr Val One-letter symbol

A

R

N

D

B

C

Q

E

z

G

L

K

M

F

P

S

T

w

Y

V

The following is a list of proteins and peptides which con',prise, principal neutralizing domains or segments thereof.

A. Recombinant Proteins Cok-aprising a Principal Neutralizing Domain 1. HIV 10 Kd fusion protein denoted Sub 1. The amino acid sequence of the HIV portion of Sub I is shown in Table 2 and the DNA sequence of the HIV portion of Sub 1 in Table 2A. The amino acid sequence of Sub I is shown in Thble 2B and the DNA sequence in Table WO 90/03984 PCT/US89/04302 6 2. HIV 18 Kd fusion protein denoted Sub 2. The amino acid sequence of the HIV portion of Sub 2 is shown in Table 3 and the DNA sequence of the HIV portion of Sub 2 in Table 3A. The entire amino acid sequence of Sub 2 is shown in Table 3B and the entire DNA sequence in Table 3C.

3. HIV 27 Kd fusion protein denoted PB1RF. The amino acid sequence of the HIV portion of PB1RF is listed in Table 4 and the DNA sequence of the HIV portion of PB1RF is listed in Table 4A. The entire amino acid sequence and DNA sequence of PB1RF are in Tables 4B and 4C, respectively.

4. HIV 28 Kd fusion protein denoted PBIMN. The amino acid sequence of the HIV portion of PB1MN is shown in Table 5 and the DNA sequence of the HIV portion of PBIMN is shown in Table 5A. The entire amino acid sequence and DNA sequence of PBlMN are shown in Tables 5B and 5C, respectively.

HIV 26 Kd fusion protein denoted PBlsc. The amino acid sequence of the HIV portion of PBlsc is listed in Table 6 and the DNA sequence of the HIV portion of PBlsc is shown in Table 6A. The entire amino acid sequence and DNA sequence of PBIsc are shown in Tables 6B and 6C, respectively.

6. HIV 26 Kd fusion protein denoted PBlwMj 2 The amino acid sequence of the HIV portion of PBlwM 2 is listed in Table 7 and the DNA sequence of the HIV portion of PBliWj 2 is shown in Table 7A. The entire amino acid sequence and DNA sequence of PBlwM32 are shown in Tables 7B and 7C, respectively.

B. Synthetic Peptides Comprising Segments of the Principal Neutralizing Domain From IIV Variants The amino acid cysteine in parentheses is added for the purpose of crosslinking to carrier proteins. Also, where the peptides have cysteines at or near both ends, these cysteines can form a disulfide bond, thus giving the peptides a loop-like configuration. For any of these peptides which do not have cysteines at or near both ends, cysteines may be added if a loop-like configuration is desired.

The loop configuration can be utilized to enhance the imiunogenic properties of the peptides. Other amino acids in parentheses are immunologically silent spacers.

Peptide 135 (from isolate HIV-IIlI): Asn Asn Thr Arg Lys Ser Ile Arg Gin Arg Gly Pro Gly Arg Ala Phe Val Thr lie Gly Lys lie Gly (Cys) WO 90/03984PIU8/40 PCr/US89/04302 Peptide 136 (from isolate H-IV-IIIB): Leu Asn Gin Ser Val Gin Ile Asn Asn Ttr Arg Lys Ser le Gly Arg Ala Phe Val TMr le Peptide 139 (from isolate HIV-RF): Asn Asn Thr Arg Lys Ser le Arg Val Hle Tyr Ala Thr Gly PepRide 141 (from isolate HIV-WMJ2): Asn Asn Val Mrg Arg Ser Len Arg Ala Phe Arg Thr Arg Gin PeRtide 142 (from isolate HIV-MN): Tyr Asn Lys Arg Lys Arg le Arg Ala Phe Tyr Thr Tbr Lys

(CYS)

Asn Cys Arg le Gly Lys 71r Lys Gin le Thr Arg Pro Asn Gin Arg Gly Pro le Gly Asn Met Gly Pro Gly le Gly (Cys) Ser le le le His le Asn le Giy Pro Gly Gly (Cys) Gly Pro Gly le Gly Peptide 143 (from isolate HIV-SC): Asn Asn Thr T11r Mg Ser le His le Gly Pro Gly Mrg Ala Phe Tyr Ala Thr Giy Asp Ile Ile Gly (Cys) Peptide 131 (from isolate HIV-IJIB3): (Tyr) Cys Thr Arg Pro Asn sn Msn TMr Mrg Lys Ser Ile Arg Ile Gin Mrg Gly Peptide 132 (from isolate HIV-IIIB3): Pro Gly Arg Ala Phe Val 7[1r le Gly Lys le Gly Msn Met Arg Gin Ala His Cys (Tyr) Peptide 134 (from isolate lT-II-lB): Gia Arg Val Ala Asp Leu Mn Gin Ser Val Ci Ile Asn Cys Thr Arg Pro Msn Mn Mn 7[1r Mg Lys Ser Ile WO 90/03984 PTU8/40 PCT/US89/04302 Peptidle 339 (from isolate HIV-FLF): le Thr Lys Cily Pro Gly RP341 (from isolate HWV-WMJ2): Leu Ser le Gly Pro Guy RP343 (from isolate HIV-SC): le His le Giy Pro Gly (from isolate HIV-IIIB):.

le Asn Cys Thr Arg Pro Arg Val Ile Tyr (Cys) Arg Ala Phe Arg (Cys) Arg Ala Plie Ty'r (Cys) Asn Asn Asn Thr Arg Lys Ser RP335 (from isolate HIV-IIIB): le Gin Arg Gly Pro RP337 (from isolate HIV-XIIB): Lys Ser le Arg le (Cys) RP77 (from isolate HIV-IIIB3): Giy Pro Giy Arg Ala Gly Arg Ala Phe (Cys) Gin Arg Gly Pro Gly Arg Ala Phe RP83 (from isolate H-IV.WM31): His le His le Gly Pro Gly Mg Ala Phe Tyr Thr Gly (Cys) RP7 (from isolate HIV-IIIB): Gin Mg Gly Pro Giy Arg Ala Phe (Cys) RP57: le Asn Cys Thr Arg Pro Ala His Cys Asn le Ser Ala His Cys Asn Ile Ser WO 90/03984 PTU8/40 PCT/US89/04302 9 (Ala Ala Ala Ala Ala Ala) Gly Pro Gly Arg (Ala Ala Ala Ala Ala Cys) RP56: le Asn Cys Thr Arg Pro RP59: Ile Gly Asp Ile Arg Gin Ala His Cys Asn Ile Ser RP342 (from isolate HIV-MN): le His Ile Gly Pro Gly Arg Ala Phe Tyr 11r (Cys) RP96 (HIV.MN related): (Cys) Gly Ile His le Gly Pro Gly Arg Ala Phe Tyr Thr (Cys) RP97 (HIV.MN related): (Ser Gly Gly) Ile His Ile Gly Pro Giy Arg Ala Phe Tyr (Gly Giy Ser Cys) RP98 (lHV-MN related): (Cys Ser Gly Gly) Ile His Ilie Gly Pro Gly Arg Ala Phe Tyr (Gly Gly Set Cys) RP99 (HIV.MN related),.

(Cys Ser Gly Gly) Ile His Ile Gly Pro Gly Arg Ala Phe Tyr (Gly Gly Set) RP100: (Ser Gly Gly) TMr Arg Lys Gly le His Ile Gly Pro Gly Arg Ala Ile Tyr (Gly Gly Ser Cys) WO 90/03984 PCr/US89/043.2 RP102: (Ser Gly Gly) 71r Arg Lys Ser le Ser Ile Gly Pro Gly Arg Ala Phe (Gly Gly Ser Cys) RP91 (MN-Hepatitis fusion): Mrg le His le Giy Pro Gly Arg Ala Phe Tyrr Gly Phe Phe Leu Leu Thr Arg le Leu 71r le Pro Gin Ser Levi Asp (Cys) RP104: (Ser Gly Gly) Ile Gly Pro Gly Arg Ala Pije Tyr Thr Thr Lys (Gly Gly Ser Cys) RP106: (Ser Gly Gly) Arg Ile His le Gly Pro Gly Mrg Ala Phe (Gly Gly Ser Cys) RP108: (Ser Gly Gly) His le Gly Pro Gly Mg Ala Phe Tyr Ala 71r Gly (Gly Gly Ser Cys) RP1O (from isolate HIV.MN): Ilie Asn Cys T-hr Mg Pro Asn Tyr Asn Lys Mrg Lys Mg Ile His Ile Gly Pro Gly Arg Ala Phe Tyr Thr 'fir Lys Asn Ile Ile Gly Thr Ile Mg Gin Ala His Cys Msn le Ser RP84 (from isolate HIV-MN): le His lie Giy Pro Gly Arg Ala Phe Tyr Thr Gly (Cys) RP144 (from isolate 7887-3):.

Msn Mn Thr Ser Mg Giy le .Ag Ile Gly Pro Gly Arg Ala Ile Leu Ala Thr Giu Mg Ile lie Gly (Cys) RP145 (from isolate 6587-7): Msn Msn Thr Mrg Lys Gly Ile His lie Gly Pro Gly Arg Ala Phe Tyr Ala Thr Gly Asp Ile lie Gly (Cys) WO 90/03984 C/U8/40 PCT/US89/04302 RP146 (from isolate CC): Asn A.%n 'Mr Lys Lys Gly lie Val Tyr TMr Ala Arg Arg le RP147 (from isolate KK261): Asn Asn Th5, Arg Lys Gy~ Ile Val Tyr Tiir Arg His Lys, lie Arg le le Gly Tyrr Val Ile Gly Gly Pro Gly Arg Ala (Cys) Gly Scr Gly Arg Lys (Cys) RP150 (from isolate ARV-2): Asn Asn T7hr Mrg Lys Ser lie Phe His Thr Thr Gly Arg le RP151 (from isolate Asn Asn Mflr Lys Lys 'Sly lie Leu Ty~ ALI Mrg Glu Lys lie Tyr Ile lie Gly Ala lie Bie Gly Giy Pro Giy Mg Ala (Cys) Giy Pro Gly Arg TMr (Cys) C. Hybrid Peptides Containing Sequences from Mora Than One Variant RP73 (from isolates HIV-IIIB, HIV-RF Lys Ser le Mrg lie Gin Mrg Gly Pro Gly Mg Val lie Tyr (Cys) RP74 (from isolates HIV-IIIB, HIV-Rr-, HIV-MN, HIV-SC): Arg lie His le Gly Pro Gly Arg Ala Phe Tyr Ala Lys Ser Ile Mrg lie Gin Mg Gly Pro Gly Mrg Val lie Tyr (Cys) RtQ (from isolates HW-lflB, H-IV.RF): Arg Bie Gla Arg 0 Paz Gly Mrg Val Ile Tyr Ala Thr

(CYS)

RP81j (from isolates HIV-IIIB, HIV-RF, HIV-WMJl, lHV-MN): Arg lie His lie Gly Pro Gly Mrg Ala Phe T'Jr Thr Gly Mrg le Gin Arg Gly Pro Gly Mrg Val lie Tyr Ala Thr (Cys) WO 90/03984 PTU8/40 PCF/US89/04302 12 RP82 (from isolates H.W-MN, HIV-VWJ): Arg He His lie Gly Pro Gly Arg Mla Phe Tfyr Tr Gly (Cys) RP88 (from isolates HIV-MN, WIV-SC): Ser le His le Gly Pro Gly Arg Ala Phe Ty~r Thir Thr Oly (Cys) RP3?.7 (from isolates HIV-IIIB3, H-IV-RF): Asn Asn Thr Arg Lys Ser lie Arg lie Thr Lys Giy Pro Gly Arg Ala Phe Val Thr le Gly Lys le Gly (Cys) RP140 (from isolates HWV-IIIB, HWV-RF): Asn Asn 71r Arg Lys Ser le Thr Lys Gly Pro Gly Arg Aa Phe Val 71r lie Gly Lys lie Gly (Cys) Peptide 64 (from isolates HIV-HIB1, HIV-RF, HIV-MN, HI V-SC): Arg Ile His lie Gly Pro Gly Arg Ala lie Phe Tyr Arg Ile. Gin Arg Gly Pro Gly Arg Val lie Tyr (Cys) Peptide 338 (iror isolates HIV-IIIB3, HIV-RF): Arg Ile Gin Arg Gly Pro Gly Arg Val le Tyr (Cys) Peptide 138 (from isolates HIV-111IB, HlV-RF): Asn Asn 71r Arg Lys Ser le Arg le GIn Arg Gly Pro Gly Arg Val le Tyr Ala TMr Giy Lys lie Giy (Cys) P rg Ile Gin Arg Gly Pro Gly Arg Val lie Tyr (Cys) D. Miscellaneous Peptide Sequences RP41: Gly Pro Gly Arg PCT/ US89/04302 WO 90/03984 RP6I: Gly (Cys Ala RP1ll: Ile RPII13: Gin Gly RP114: Arg Pro RP1I16: Gly Giy RP120: Ser Pro Gly Arg (Ala Ala Ala Ala Ala Cys) Ala Ala Ala) Gly Ala Ala Ala Cys) Pro Gly Arg Ala Phe (Ala Ala Gin Arg Gly Pro Gly 2ys) Arg Gly Pro Gly Arg Gin Arg Gin Mg Gly Pro Gly le Arg Gly Pro Gly Mrg Gin Pro Gly Arg (Cys) Gly Pro Gly Arg Gly Pro Gly AMg Gly Pro Gly Arg Gly Gly Arg (Cys) Pro Gly Mg Ala Phe Gly Pro Gly Mrg Ala Phe Gly Pro Arg Ala, Phe (Cys) le rg Ile Gly Pro Gly Mrg Ala Phe Tyr Thr (Cys) RP12Ic: (Cys) Gly RP122c: (Cys) Ile RP3 23c: (Cys) His Pro Gly Mrg (Cys) Gly Pro Giy Mrg Ala (Cys) Ile Gly Pro Gly Mrg Ala Phe (Cys) WO 90/03984 PC/US89/04302 14 The proteins and peptides exemplifying the subject invention can be made by well-knovn synthesis procedures. Alternatively, these entities can be made by use of recombinant DNA procedures. Such recombinant DNA procedures are disclosed herein since they were, in fact, the procedures initially utilized to obtain the novel proteins and peptides of the invention. However, once these entities were prepared and their molecules sequenced, it is apparent to a person skilled in the art that the preferred method for making them would now be by chemical synthesis means. For example, there are available automated machines which can readily make proteins and peptides of the molecular sizes disclosed herein.

In the recombinant DNA procedures for making some of the proteins and peptides of the invention, an expression vector plasmid denoted pREV2.2 was used. This plasmid was initially constructed fromn a plasmid denoted pBG1.

Plasmid pBGl is deposited in the E. coli host MS371 with the Northern Regional Research Laboratory (NRRL, U.S. Department of Agriculture, Peoria, Illinois, USA). It is in the permanent collection of this repository. E coii MS371(pBG1), NRRI B-15904, was deposited on November 1, 1984. E. coli MS371, NRRL B-15129 is now available to the public.

Plasmid pREV2.2 was deposited in the E. coli JM103 host with NRRL on July 30, 1986. E coli JM103(pREV2.2) received the accession number NRRL B-18091. NRRL B-15904 and NRRL B-18091 will be available, without restrictions, to the public upon the grant of a patent which discloses them.

Other E. coli strains, disclosed herein, were deposited as follows: E. coli SG20251, NRRL B-15918, was deposited on December 12, 1984.

E. coli CAG629(pKH1), NRRL B-18095, was deposited on July 30, 1986.

This latter deposit can be subjected to standard techniques to separate the plasmid from the host cell, and, thus, use the host E. coli CAG629 as disclosed herein.

The subject cultures have been deposited under conditions that assure that access to the cultures will be available during the pendency of the patent application disclosing them to one determined by the Commissioner of Patents and Trademarks to be entitled thereto under 37 CFR 1.14 and 35 USC 122. The deposits are available as required by foreign patent laws in countries wherein counterparts of the subject application, or its progeny, are filed. However, it should be understood that the availability of a deposit does not constitute a license to practice the subject invention in derogation of patent rights granted by governmental action.

Further, the subject culture deposits will be stored and made avaiable to the public in accord with the provisions of the Budapest Treaty for the Deposit of Microorganisms, they will be stored with all the care necessary to keep them viable and uncontaminated for a period of at least five years after the most recent request for the furnishing of a sample of the deposits, and in any case, for a WO 90/03984 PCT/US89/04302 period of at least 30 (thirty) years after the date of deposit or for the enforceable life of any patent which may issue disclosing the cultures. The depositor acknowledges the duty to replace the deposits should the depository be unable to furnish a sample when requested, due to the condition of the deposits. All restrictions on the availability to the public of the subject culture deposits will be irrevocably removed upon the granting of a patent disclosing them.

The novel HIV proteins and peptides of the subject invention can be expressed in Saccharomvces cerevisiae using plasmids containing the inducible galactose promoter from this organism (Broach, Y. Li, LC. Wu, and M. Jayaram [1983] in Experimental Manipulation of Gene Expression, p. 83, ed. M. Inouye, Academic Press). These plasmids are called YEp51 and YEp52 (Broach, J.R. et al [1983]) and contain the E. coli origin of replication, the gene for fl-lactamase, the yeast LEU2 gene, the 2 pm origin of replication and the 2 pm circle REP3 locus. Recombinant gene expression is driven by the yeast GAL10 gene promoter.

Yeast promoters such as galactose and alcohol dehydrogenase (Bennetzen, J.L. and B.D. Hall [1982] J. Biol. Chem. 257:3018; Ammerer, G. [1983] in Methods in Enzymology Vol. 101, p. 192), phosphoglycerate kinase (Derynck, R.A. Hitzeman, P.W. Gray, D.V. Goeddel [19831 in Experimental Manipulation of Gene Expression, p. 247, ed. M. Inouye, Academic Press), triose phosphate isomerase (Alber, T. and G. Kawasaki [1982] J. Molec. and Applied Genet. 1:419), or enolase (Innes, M.A. et al. [1985] Science 226:21) can be used.

The genes disclosed herein can be expressed in simian cells. When the genes encoding these proteins are cloned into one of the plasmids as described in Okayama and Berg (Okayama, H. and P. Berg [1983] Molec. and Cell. Biol. 3:280) and references therein, or COS cells transformed with these plasmids, synthesis of HIV proteins can be detected immunologically.

Other mammalian cell gene expression/protein production systems can be used. Examples of other such systems are the vaccinia virus expression system (Moss, B. [19851 Immunology Today 6:243; Chakrabarti, K. Brechling, B. Moss [1985] Molec. and Cell. Biol. 5:3403) and the vectors derived from murine retroviruses (Mulligan, R.C. 11983] in Experimental Manipulation of Gene Expression, p. 155, ed. M. Inouye, Academic Press).

The HIV proteins and peptides of the subject invention can be chemically synthesized by solid phase peptide synthetic techniques such as BOC and FMOC (Merrifield, R.B. 119631 J. Amer. Chem.

Soc. 85:2149; Chang, C. and J. Meienhofer [1978] Int. J. Peptide Protein Res. 11:246).

As is well known in the art, the amino acid sequence of a protein is determined by the nucleotide sequence of the DNA. Because of the redundancy of the genetic code, more than one coding nucleotide triplet (codon) can be used for most of the amino acids used to make proteins, different nucleotide sequences can code for a particular amino acid. Thus, the genetic code can be depicted as follows: WO 90/03984 WO 90/03984PCT/US89/04302 16 Phenylalanine (Phe) ITrK Histidine (His) CAK Leucine (Leu) XTY Glutamine (Gin) CAJ Isoleucine (lie) ATM Asparagine (Asn) AAK Methionine (Met) ATO Lysine (Lys) AMJ Valine (Val) GTL Aspartic acid (Asp) GAK Serine (Ser) QRS Glutamic acid (Glu) GAJ Praline (Pro) CCL Cysteine (Cys) TGK Threonine (Thr) ACL Tryptophan (Trp) TG Alanine (Ala) GCL Arginine (Arg) WGZ Tyrosine (Tyr) TAX Glycine (Gly) GGL Termination signal TMJ Termination signal TGA Key: Each 3-letter deoxynucleotide triplet corresponds to a trinucleotide of niRNA, having a on the left and a 3'-end on the right. All DNA sequences given herein are those of the strand whose sequence corresponds to the niRNA seque.-Zz!, with thymine substituted for uracil. The letters stand for the purine or pyrimidine bases forming the deoxynucleotide sequence.

A adenine O guanine C cYtosine, T thynine X T or C if Y is A or G X C if Y is C or T Y G, C or T if X is C Y A or G if X is T W C or Aif Zis Aor G W C if Z is C or T Z A, C or T if W is C Z A or G if W is A OR TC if S is A, G3, C or T, alternatively QR =AGif Sis Tor C J =A or G K =T or C L T, C or G M C or T WO 90/03984 PCT/US89/04302 17 The above shows that the novel amino acid sequences of the HIV proteins and peptides of the subject invention can be prepared by nucleotide sequences other than those disclosed herein.

Functionally equivalent nucleotide sequences encoding the novel amino acid sequences of these HIV proteins and peptides, or fragments thereof having HIV antigenic or immunogenic or therapeutic activity, can be prepared by known synthetic procedures. Accordingly, the subject invention includes such functionally equivalent nucleotide sequences.

Thus the scope of the subject invention includes not only the specific nucleotide sequences depicted herein, but also all equivalent nucleotide sequences coding for molecules with substantially the same HIV antigenic or immunogenic or therapeutic activity.

Further, the scope of the subject invention is intended to cover not only the specific amino acid sequences disclosed, but also similar sequences coding for proteins or protein fragments having comparable ability to induce the formation of and/or bind to specific HIV antibodies possessing the properties of virus neutralization.

The term "equivalent" is being used in its ordinary patent usage here as denoting a nucleotide sequence which performs substantially as the nucleotide sequence identified herein to produce molecules with substantially the same HIV antigenic or immunogenic or therapeutic activity in essentially the same kind of hosts. Within this definition are subfragments which have HIV antigenic or immunogenic or therapeutic activity.

As disclosed above, it is well within the skill of those in the genetic engineering art to use the nucleotide sequences encoding HIV antigenic or immunogenic or therapeutic activity of the subject invention to produce HIV proteins via microbial processes. Fusing the sequences into an expression vector and transforming or transfecting into hosts, either eukaryotic (yeast or mammalian cells) or prokaryotic (bacterial cells), are standard procedures used in producing other well-known proteins, e.g., insulin, interferons, human growth hormone, IL-1, IL-2, and the like. Similar procedures, or obvious modifications thereof, can be employed to prepare HIV proteins or peptides by microbial means or tissue-culture technology in accord with the subject invention.

The nucleotide sequences disclosed herein can be prepared by a "gene machine" by procedures well known in the art. This is possible because of the disclosure of the nucleotide sequence.

The restriction enzymes disclosed can be purchased from Bethesda Research Laboratories, Gaithersburg, MD, or New England Biolabs, Beverly, MA. The enzymes are used according to the instructions provided by the supplier.

The various methods employed in the preparation of the plasmids and transformation of host organisms are well known in the art. These procedures are all described in Manialis, E.F. Fritsch, and J. Sambrook (1982) Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory, New York. Thus, it is within the skill of those in the genetic engineering art to extract DNA from WO 90/03984 PCT/US89/04302 18 microbial cells, perform restriction enzyme digestions, electrophorese DNA fragments, tail and anneal plasmid and insert DNA, ligate DNA, transform cells, E. coli cells, prepare plasmid DNA, electrophorese proteins, and sequence DNA.

Immunochemical assays employing the HIV proteins or peptides of the invention can take a variety of forms. One preferred type is a liquid phase assay wherein the HIV antigen and the sample to be tested are mixed and allowed to form immune complexes in solution which are then detected by a variety of methods. Another preferred type is a solid phase immunometric assay. In solid phase assays, an HIV protein or peptide is immobilized on a solid phase to form an antigenimmunoadsorbent. The immunoadsorbent is incubated with the sample to be tested. After an appropriate incubation period, the immunoadsorbent is separated from the sample, and labeled anti- (human IgG) antibody is used to detect human anti-HIV antibody bound to the immunoadsorbent.

The amount of label associated with the immunoadsorbent can be compared to positive and negative controls to assess the presence or absence of anti-HIV antibody.

The immunoadsorbent can be prepared by adsorbing or coupling a purified HIV protein or peptide to a solid phase. Various solid phases can be used, such as beads formed of glass, polystyrene, polypropylene, dextran or other material. Other suitable solid phases include tubes or plates formed from or coated with these materials.

The HIV proteins or peptides can be either covalently or non-covalently bound to the solid phase by techniques such as covalent bonding via an amide or ester linkage or adsorption. After the HIV protein or peptide is affixed to the solid phase, the solid phase can be post-coated with an animal protein, 3% fish gelatin. This provides a blocking protein which reduces nonspecific adsorption of protein to the immunoadsorbent surface.

The immunoadsorbent is then incubated with the sample to be tested for anti-HIV antibody.

In blood screening, blood plasma or serum is used. The plasma or serum is diluted with normal animal plasma or serum. The diluent plasma or serum is derived from the same animal species that is the source of the anti-(human IgG) antibody. The preferred anti-(human IgG) antibody is goat anti-(human IgG) antibody. Thus, in the preferred format, the diluent would be goat serum or plasma.

The conditions of incubation, pH and empc. iture, and the duration of incubation are not crucial. These parameters can be optimized by routine experimentation. Generally, the incubation will be run for 1-2 hr at about 45 0 C in a buffer of pH 7-8.

After incubation, the immunoadsorbent and the sample are separated. Separation can be accomplished by any conventional separation technique such as sedimentation or centrifugation. The immunoadsorbent then may be washed free of sample to eliminate any interfering substance.

The immunoadsorbent is incubated with the labeled anti-(human IgG) antibody (tracer) to detect human antibody bound thereto. Generally the immunoadsorbent is incubated with a solution WO 90/03984 PC'T/US8904302 19 of the labeled anti-(human IgG) antibody which contains a small amount (about of the serum or plasma of the animal species which serves as the source of the anti-(human IgG) antibody. Anti- (human IgG) antibody can be obtained from any animal source. However, goat anti-(human IgG) antibody is preferred. The anti-(human IgG) antibody can be an antibody against the Fc fragment of human IgG, for example, goat anti-(human IgG) Fc antibody.

The anti-(human IgG) antibody or anti-(human IgG) Fc can be labeleu with a radioactive material such as 125; labeled with an optical label, such as a fluorescent material; or labeled with an enzyme such as horseradish peroxidase. The anti-human antibody can also be biotinylated and labeled avidin used to detect its binding to the immunoadsorbent.

After incubation with the labeled antibody, the immunoadsorbent is separated from the solution and the label associated with the immunoadsorbent is evaluated. Depending upon the choice of label, the evaluation can be done in a variety of ways. The label may be detected by a gamma counter if the label is a radioactive gamma emitter, or by a fluorimeter, if the label is a fluorescent material. In the case of an enzyme, label detection may be done colorimetrically employing a substrate for the enzyme.

The amount of label associated with the immunoadsorbent is compared with positive and negative controls in order to determine the presence of anti-HIV antibody. The controls are generally run concomitantly with the sample to be tested. A positive control is a serum containing antibody against HIV; a negative control is a serum from healthy individuals which does not contain antibody against HIV.

For convenience and standardization, reagents for the performance of the immunometric assay can be assembled in assay kits. A kit for screening blood, for example, can include: an immunoadsorbent, a polystyrene bead coated with an HIV protein or peptide; a diluent for the serum or plasma sample, e.g. normal goat serum or plasma; an anti-(human IgG) antibody, goat anti-(human IgG) antibody in buffered, aqueous solution containing about 1% goat serum or plasma; a positive control, serum containing antibody against at least one of the novel HIV proteins or peptides; and a negative control, pooled sera from healthy individuals which does not contain antibody against at least one of the novel HIV proteins or peptides.

If the label is an enzyme, an additional element of the kit can be the substrate for the enzyme.

Another type of assay for anti-HIV antibody is an antigen sandwich assay. In this assay, a labeled HIV protein or peptide is used in place of anti-(human IgG) antibody to detect anti-HIV antibody bound to the immunoadsorbent. The assay is based in principle on the bivalency of antibody molecules. One binding site of the antibody binds the antigen affixed to the solid phase; the second WO 90/03984 PC/US89/0 4302 is available for binding the labeled antigen. The assay procedure is essentially the same as described for the immunometric assay except that after incubation with the sample, the immunoadsorbent is incubated with a solution of labeled HIV protein or peptide. HIV proteins or peptides can be labeled with radioisotope, an enzyme, etc. for this type of assay.

In a third format, the bacterial protein, protein A, which binds the Fc segment of an IgG molecule without interfering with the antigen-antibody interaction can be used as the labeled tracer to detect anti-HIV antibody adsorbed to the immunoadsorbent. Protein A can be readily labeled with a radioisotope, enzyme, or other detectaole species.

Immunochemical assays employing an HIV protein or peptide have several advantages over those employing a whole (or disrupted) virus. Assays based upon an HIV protein or peptide will alleviate the concern over growing large quantities of infectious virus and the inherent variability associated with cell culturing and virus production. Further, the assay will help mitigate the reil or perceived fear of contracting AIDS by technicians in hospitals, clinics and blood banks who perform the test.

Immunochemical assays employing recombinant envelope proteins from multiple viral variants have additional advantages over proteins from a single HIV variant. Assays incorporating protein sequences from multiple variants are more likely to accurately survey antibodies in the human population infected with diverse HIV variants. Also, solid phase enzyme-linked immunosorbent assay (ELISA) utilizing different HIV variant proteins would allow determination of prevalen; serotypes in different geographic locations. This determination has not been possible until now as no available antibody detection kit utilizes mor2 than one HIV variant.

Another use of recombinant proteins from HIV variants is to elicit variant-specific antiscra in test animals. This antiserum would provide a reagent to identify which viral variant infected an individual. Currently, "virus typing" can only be done by viral gene cloning and sequencing. Binding of variant-specific serum to a patient viral isolate would provide a means of rapid detection not currently available. For example, sera raised to the proteins denoted PB1 1 ln, PBR-, PBIMN, PBlsc, and PBwhMJ2 can be used to screen viral isolates from patients to determine which HIV variant the clinical isolate most closely resembles. This "screening" can be done by a variety of known antibodyantigen binding techniques.

Vaccines comprising one or more of the HIV proteins or peptides, disclosed herein, and variants thereof having antigenic properties, can be prepared by procedures well known in the art.

For example, such vaccines can be prepared as injectables, liquid solutions or suspensions. Solid forms for solution in, or suspension in, a liquid prior to injection also can be prepared. Optionally, the preparation also can be emulsified. The active antigenic ingredient or ingredients can be mixed with excipients which are pharmaceutically acceptable and compatible with the active ingredient.

WO 90/03984 PC/US89/04302 21 Examples of suitable excipients are water, saline, dextrose, glycerol, ethanol, or the like, and combinations thereof. in addition, if desired, the vaccine can contain minor amounts of auxiliary substances such as wetting or emulsifying agents, pH buffering agents, or adjuvants such as aluminum hydroxide or muramyl dipeptide or variations thereof. In the case of peptides, coupling to larger molecules such as KLH sometimes enhances immunogenicity. The vaccines are conventionally administered parenterally, by injection, for example, either subcutaneously or intramuscularly.

Additional formulations which are suitable for other modes of administration include suppositories and, in some cases, oral formulations. For suppositories, traditional binders and carriers include, for example, polyalkalene glycols or triglycerides. Suppositories can be formed from mixtures containing the active ingredient in the range of about 0.5% to abcut 10%, preferably about 1 to about Oral formulations can include such normally employed excipients as, for example, pharmaceutical grades of mannitol, lactose, starch, magnesium stearate, sodium saccharine, cellulose, magnesium carbonate, and the like. These compositions can take the form of solutions, suspensions, tablets, pills, capsules, sustained release formulations or powders and contain from about 10% to about 95% of active ingredient, preferably from about 25% to about The compounds can be formulated into the vaccine as neutral or salt forms. Pharmaceutically acceptable salts include the acid addition salts (f.omed with the free amino groups of the peptide) and which are formed with inorganic acids such as, for example, hydrochloric or phosphoric acids, or such organic acids as acetic, oxalic, tartaric, mandelic, and the !ike. Salts fr.ianed with the free carboxyl groups can also be derived from inorganic bases such as, for example, sodium, potassium, ammonium, calcium, or ferric hydroxides, and such organic bases as isopropylamine, trimethylamine, 2-ethylamino ethanol, histidine, procaine, and the like. A vaccine composition may include peptides containing T helper cell epitopes in combination with protein fragments containing the principal neutralizing domain. Several of these epitopes have been mapped within the HIV envelope, and these regions have been shown to stimulate proliferation and lymphokine release from lymphocytes. Providing both of these epitopes in a vaccine may result in the stimulation of both the humoral and the cellular immune responses.

Alternatively, a vaccine composition may include a compound which functions to increase the general immune response. One such compound is interleukin-2 (IL-2) which has been reported to enhance immunogenicity by general immune stimulation (Nunberg et al. (1988] In New Chemical and Genetic Approaches to Vaccination, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY). IL- 2 may be coupled with an HIV peptide or protein comprising the PND to enhance the efficacy of vaccination.

The vaccines are administered in a manner compatible with the dosage formulation, and in such amount as will be therape.utically effective and immunogenic. The quantity to be administered WO 90/03984 PC/US9/04302 22 depends on the subject to be treated, capacity of the subject's immune system to synthesize antibodies, and the degree of protection desired. Precise amounts of active ingredient required to be administered depend on the judgment of the practitioner and are peculiar to each individual. However, suitable dosage ranges are of the order of about several hundred micrograms active ingredient per individual.

Suitable regimes for initial administration and booster shots are also variable, but are typified by an initial administration followed in one or two week intervals by a subsequent injection or other administration.

HIV is known to undergo amino acid sequence variation, particularly in the envelope gene (Starcich, B.R. [19861 Cell 45:637-648; Hahn, B.H. et al. [1986] Science 232:1548-1553). Over 100 variants have been analyzed by molecular cloning and restriction enzyme recognition analysis, and several of these have been analyzed by nucleotide sequencing. Some of these are the HIV isolates known as RF (Popovic, M. et al. [1984] Science 224:497-500), WMJ-1 (Hahn, B.H. et al. (19861 Science 232:1548-1553), LAV (Wain-Hobson, S. et al. [1985] Cell 40:9-17), and ARV-2 (Sarchez- Pescador, R. et al. [1985] Science 227:484-492). HIV peptides from different viral isolates can be used in vaccine preparations to protect against infections by different HIV isolates. Further, a vaccine preparation can be made using more than one envelope protein fragment corresponding to the principal neutrilizing domain of more than one HIV isolate to provide immunity and thus give better protection against AIDS. Alternatively, the vaccine preparation can be made using a single protein fragment that is comprised of a tandem arrangement of principal neutralizing epitopes from more than one HIV isolate. By identifying the principal neutralizing domain of HIV, this polypeptide region can be applied to formulate valuable vaccine, diagnostic, and therapeutic reagents.

Antibodies to recombinant peptides disclosed herein are useful as therapeutic and prophylactic reagents. The generation of polyclonal or monoclonal antibodies capable of neutralizing a variety of HIV variants could be used to reduce the incidence of accidental infection and treat HIV infected people that are immuno-compromised. Additionally, immunization regimens may elicit polyclonal sera capable of broadly neutralizing several variants of HIV. The ability to neutralize multiple HIV variants is termed broadly neutralizing antibody. Broadly neutralizing antibody may neutralize two or more HIV variants or all HIV variants. Therefore, a mixture of broadly neutralizing antibodies that neutralize different groups of HIV variants would be useful fordiagnosis, prophylaxis, and therapy of

AIDS.

It is surprising and advantageous that immunization with peptides from five HIV variants would yield sera capable of neutralizing more than these five HIV variants when immunization with two does not. Example 22 shows that immunization with five peptides elicits broadly neutralizing sera.

Broadly neutralizing sera may also be generated if several sequences from the hypervariable region of diverse HIV variants are presented as a single synthetic peptide. Additionally, one may licit this WO 90/03984 PP/US89/04302 23 broadly neutralizing sera by reimmuniaation of animals primed with RP136 or equivalent proteins with peptides containing only the conserved amkio acids within this hypervariable region. These immunization regimens would be useful for vaccination and for deriving antibodies useful as therapeuic agents.

Polyvalent immune globulin for use in passive immunization can be prepared by immunization of horses or by pooling immune human sera and fractionation of the IgG component from plasma or sera. Human or mouse monoclonal antibody producing cell lines may be prepared by standard transformatio n s ~,tidoma technology (Methods in Enzvmolov Vol. 121, Sections I and 1I (1986] eds. JJ. Latgone and H.V. Vunakis, Academic Press). HIV monoclonal antibody can be prepared in accord with e proce-ures disclosed by Matsushita et al, (Matsushita et al. [19881 Journal of Virology 62(6):2107-2114). Sinice, for the most part, monoclonal antibodies are produced in species other than humans, they are often immunogenic to humans. In order to successfully use these monoclonal antibodies in the treatment of humans, it may be necessary to create a chimeric antibody molecule wherein the portion of the polypeptide involved with ligand binding (the variable region) is derived from one species, and the portion involved with providing structural stability and other biological functions (the constant region) is derived from a human antibody. Methods for producing chimeric antibodies in which the variable domain is derived from one host and the constant domain is derived from a second host are well known to those skilled in the art. See, for example, Neuberger et al., WO Publication No. 86/01533, priority 9/3/84; Morrison et al., EP Publication No. 0 173 494, priority 8/27/84. An alternative method, in which an antibody is produced by replacing only the complementarity determining regions (CDRs) of the variable region with the CDRs from an immunoglobulin of the desired antigenic specificity, is described by Winter (GB Publication No. 2 188 638, priority 3/27/86). Murine monoclonals can he made compatible with human therapeutic use by producing an antibody containing a human Fc portion (Morrison, S.L [1985] Science 229:1202-1207).

Established procedures would allow construction, expression, and purification of such a hybrid monoclonal antibody. Regimens for administering immune globulin therapeutically have previously been used for a number of infectious diseases.

As used herein, the term "antibody" is meant to encompass monoclonal or polyclonal antibodies, whole, intact antibodies or antibody fragments having the immunological activity of the whole antibody. Also encompassed within the term "antibody' are chimeric antibodies having the variable and constant regions from different host species, or those wherein only the CDRs are replaced.

For treatment of HIV infection, compositions comprising antibodies may be administered to an individual or animal in need of treatment. Alternatively, the HIV antigens described here may be administered in order to stimulate the recipient's own immune response. When treating with an HIV antigen, a single antigen may be administered or, preferably, a broadly neutralizing antigen or mixture WO 90/03984 PCT/US89/04302 24 of antigens may be administered. Such compositions are described in detail in the examples which follow.

The ability to modify peptides made by organic synthesis can be advantageous for diagnostic, therapeutic, and prophylactic use by improving efficiency of immobilizatiun, increasing protein stability, increasing immunogenicity, altering immunogenicity, reducing toxicity, or allowing multiple variations simultaneously. For example, peptides can be modified to increase or decrease net charge by modification of amino or carboxyl groups (carbamylation, trifluoroacetylation or succinylation of amino groups; acetylation of carboxyl groups). Peptides can be made more stable by, for example, inclusion of D-amino acids or circularization of the peptide. Reductive state of peptides can be altered by, for example, sulfonation of cystinyl groups. Peptides can also be modified covalently or non-covalently with non-proteinaceous materials such as lipids or carbohydrates to enhance immunogenicity or solubility. Polyethylene glycol can be used to enhance solubility. The subject invention includes all such chemical modifications of the proteins and peptides disclosed herein so long as the modified protein and/or peptide retains substantially all the antigenic/immunogenic properties of the parent compound.

Peptides can also be modified to contain antigenic properties of more than one viral variant.

This has been done, for example, with Foot and Mouth Disease virus.

Foot and Mouth Disease virus is similar to HIV in that multiple variants exist and immunization with one variant does not lead to protection against other variants. The reai utility of peptides as immunogens is demonstrated by eliciting immunity to more than one variant by modification of the peptide to possess properties of both natural variants. When such a modified variant was used to immunize test animals, they were protected against both Foot and Mouth virus strains A10 and A12 (Brown, F. in Virus Vaccines, ed. G. Dreesman, J. Bronson, R. Kennedy, pp. 49- 54 11985]).

HIV peptides or proteins containing a PND epitope can also be coupled with or incorporated into an unrelated virus particle, a replicating virus, or other microorganism in order 1o enhance immunogenicity. The HIV epitope may be genetically or chemically attached to the viral parith §r microorganism or an immunogenic portion or component thereof. Antigenic epitopes have been attached to viral proteins or particles to enhance the immune response. For example, the VP6 capsid protein of rotavirus has been used as an immunologic carrier protein for an epitope of interest either in the monomeric form or as oligomers of VP6 in the form of particles (EP Publication No. 0 259 149). Similarly, Evans et al. (1989, Nature 339:385) have constructed chimaeras of the poliovirus capsid protein and an epitope of HIV gp41 to enhance immunogenicity of the HIV epitope. Foreign antigenic determinants have also been expressed and presented by bacterial cells. A Salmonella strain expressing a cloned Salmonella flagellin gene, into which was inserted an epitope of either cholera WO 90/03984 PCr/US89/04302 toxin or hepatitis B surface antigen, was reported to elicit both cellular and humoral responses to the inserted epitopes (Newton et al. [1989] Science 244:70-72; and Wu et al. 11989] Proc. Natl. Acad. Sci.

86:4726-4730).

Example 18 shows that a peptide containing amino acid sequences from two HIV variants can block virus neutralization activity of two virus specific neutralizing antisera. This suggests that a peptide or protein containing sequences of two or more HIV variants can elicit an immune response etfective against two or more HIV variants.

Example 19 shows that co-immunization with envelope proteins from two HIV isolates elicits an immune response capable of neutralizing two HIV isolates. This suggests that co-immunization with proteins from two or more HIV variants can elicit an immune response effective against two or more HIV variants.

Following are examples which illustrate the process of the invention, including the best mode.

These examples should not be construed as limiting. All solvent mixture proportions are by volume unless otherwise noted.

Example 1 Construction of plasmid pREV2.2 The pREV2.2 plasmid expression vector was constructed from plasmid pBGl. Plasmid pBGl can be isolated from its E. coli host by well known procedures, using cleared lysate-isopycnic density gradient procedures, and the like. Like pBG1, pREV2.2 expresses inserted genes behind the E coli promoter. The differences between pBO1 and pREV2.2 are the following: I. pREV2.2 lacks a functional replication of plasmid (rop) protein.

2. pREV2.2 has the trA transcription terminator inserted into the AatlI site. This sequence insures transcription termination of over-expressed genes.

3. pREV2.2 has genes to provide resistance to ampicillin and chloramphenicol, whereas pBG1 provides resistance only to ampicillin.

4. pREV2.2 contains a sequence encoding sites for several restriction endonucleases.

The following procedures were used to make each of the four changes listed above: la. 5 pg of plasmid pBG1 was restricted with Ndel, wl'ch gives two fragments of .Approximately 2160 and 3440 base pairs.

lb. 0.1 pg of DNA from the digestion mixture, after inactivation of the Ndel, was treated with T4 DNA ligase under conditions that favor intramolecular ligation (200 Pi reaction volume using standard T4 ligase reaction conditions [New England Biolabs, Beverly, Intramolecular ligation of ,he 3440 base pair fragment gave an ampicillin resistant plasmid. The ligation mixture was transformed into the recipient WO 90/03984 pCr/US89/04l302 26 strain E. coli JM103 (available from New England Biolabs) and ampicillin resistant clones were selected by standard procedures.

Ic. The product plasmid, pBG1AN, where the 2160 base pair Ndel fragment is deleted from pBG1, was selected by preparing plasmid from ampicillin iresistant clones and determining the restriction digestion patterns with Ndel and Sail (product fragments approximately 1790 and 1650). This deletion inactivates the ro gene that controls plasmid replication.

2a. 5 pg of pBG1 N was then digested with EcoRI and Bell and the larger fragment, approximately 2455 base pairs, was isolated.

2b. A synthetic double stranded fragment was prepared by the procedure of Itakura et al.

(Itakura, IK, J.J. Rossi, and R.B. Wallace [1984] Ann. Rev. Biochem. 53:323-356, and references therein) with the following structure:

GATCAAGCTCTOCAGTCGACGCAT

3' ITCGAAGACGTCAGCTGCGTACGCC GCGGATCCGGTACCCGGGAGCTCG 3' TAGGCCATGGGCCCTCGAC1CITAA This fragment has Bell and EcoRI sticky ends and contains recognition sequences for several restriction endonucleases.

2c. 0.1 pg of the 2455 base pair EcoRI-Bcll fragment and 0.01 pg of the synthetic fragment were joined with T4 DNA ligase and competent cells of strain JM103 were transformed. Cells harboring the recombinant plasmid, where the synthetic fragment was inserted into pBG1AN between the Bell and EcoRI sites, were selected by digestion of the plasmid with HpaI and EcoRI. The diagnostic fragment sizes are approximately 2355 and 200 base pairs. This plasmid is called pREV1.

2d. 5 pg of pREVI were digested with AatlI, which cleaves uniquely.

2e. The following double-stranded fragment was synthesized:

CGGTACCAGCCCGCCTAATG

3' TGCAGCCATGGTCGGGCGGA AGCGGGCITI'rI'TGACGT3' TTACTCGCCCGAAAAAAAAC This fragment has AatllI sticky ends and contains the trnA transcription termination sequence.

2f. 0.1 pg of AatII digested pREV1 was ligated with 0.01 pg of the synthetic fragment in a volume of 20 ul using T4 DNA ligase.

WO 90/039V4 PTU8/40 PCT/US89/04302 27 2g. Cells of strain JM103, made competent, were transformed and ampicillin resistant clones selected.

2h. Using a Kpnl, R double restriction digest of plasmid isolated from selected colon ies, a cell containing the correct construction was isolated. The sizes of the KinI EoRI generated fragments are approximately 2475 and '80 base pairs. This plasmid is called pREVlTT and contains the IrpA transcription terminator.

3a. 5 ug of pREV1T, prepared as disclosed above (by standard methods) was cleaved with NdeI and Xxnnl and the approximately 850 base pair fragment was isolated.

3b. 5 ug of plasinid pBR325 (BRL, Gaithersburg, MD), which contains the genes conferring resistance to chlorarnphenicol as well as to amnpicillin and tetracycline, was cleaved with Bell and the ends blunted with Kienow polymerase and dexoynucleotides.

After inactivating the enzyme, the mixture was treated with Ndel and the approimately 3185 base pair fragment was isolated. This fragment contains the genes for chloamphenicol and amp~cillin resistance and the origin of replication.

is3c. 0.1 pg of the Ndel-XmnI fragment from pREV17T and the NdeI-Bc I fragment from pBR32S were ligated in 20 p1 with T4 DNA ligase and the mixture u;ied to transform competent Cells of strain JM1O3. Cells ,,esistant to both ampicillin and chioramphenicol were selected.

3d. Using an 1 RI and Ndel double digest of plasmid from selected clones, a plasmid wvas selected giving fragment sizes of approximately 2480, 1145, and 410 base pairs.

This is called 'ii pRPVlTr/chl and has genes for resistance to both ampicillin and chlorampher,%il.

4a. The following douale-stranded fragment was synthesized: Miul EcoRV rla! 'Lainlll Sall 1-indlll Sinal 5' CGAACGCGTGGC(ATATCATCGATGG- ATCcGTCGACAAGCTTCCCGGGAGCT 3' 3' GC'FrGCGCACCGGCATAUTAGCTAC- CTAGGCAGCrCYITCGAAGGGCCC This fragment, with a blunt end and an SstI sticky end, contains recognition sequences for several restriction enzyme sites.

4b. 5 pg of pREV17T/chi was cleaved with N~rul (which cleavss ab-out 20 nucleotides from the Bcll site) and Sstl (which cleaves within the multiple cloning site). The larger fragment, approximately 3990 base pairs, was isolated from an agarose gel.

4c. 0.1 pg of the NruI-Sstl fragment fromn pREV1'I~ichl and 0.01 ug of the synthetic fragment were treateu vilh T4 DNA ligase in a volume of 20 p1.

WO 90/03984 PcFIUS89/04302 28 4d. This mixture was transformed into strain JM103 and ampicillin resistant clones were selected.

4e. Plasmid was purified from several clones and screened by digestion with Mlul or Clal.

Recombinant clones with the new multiple cloning site will give one fragment when digested with either of these enzymes, because each cleaves the plasmid once.

4f. The sequence of the multiple cloning site was verified. This was done by restricting the plasmid with Hpal and PvuIl and isolating the 1395 base pair fragment, cloning it into the Smal site of mpl8 and sequencing it by dideoxynucleotide sequencing using standard methods.

4g. This plasmid is called pREV2.2.

Example 2 Construction of the bacterial expression vector pREV2.1 Plasmid pREV2.1 was constructed using plasmid pREV2.2 and a synthetic oligonucleotide.

The resulting plasmid was used to construct pPBl-Sub 1 and pPB1-Sub 2.

An example of how to construct pREV2.1 is as follows: 1. Plasmid pREV2.2 is cleaved with restriction enzymes Nrul and BamHI and the 4 Kb fragment is isolated from an agarose gel.

2. The following double-stranded oligonucleotide is synthesized: 5' CGAACGCGTOGTCCGATATCATCGATG 3' 3' GCTGCGCACCAGGCTATAGTAGCTACCTAG 3. The fragments from 1 and 2 are ligated in 20,ul using T4 DNA ligase, transformed into competent E. coli cells and chloramphenicol resistant colonies are isolated.

4. Plasmid clones are identified that contain the oligonucleotide from 2. spanning the region from the NruI site to the BamHI site and recreating these two restriction sites.

This plasmid is termed pREV2.1.

Example 3 Construction of and expression from plasmid pPB1-Sub 1 Plasmid pPB1-Sub 1, which contains approximately 165 base pairs (bp) of DNA encoding essentially the HIV env gene from the Pvull site to the Dral site, and from which is synthesized an approximately 12 Kd fusion protein containing this portion of the gpl20 envelope protein can be constructed as follows: 1. Restricting plasmid pPBlImg with Mlul and Dral and isolating the approximately 165 bp fragment.

WO 90/03984 PrU8/40 PCr/US89/04302 29 2. Restricting plasmid pREV2.1 with Miul and Sinai and isolating the large fragment, approximately 4 Kd, from an agarose gel.

3. Ligating the fragn.,.ent pr epared in 2. with the pREV2.1 fragment in a volume of pt using T4 DNA ligase, transforming the ligation mixture into competent cell strain CAG629, and selecting ampicillin-resistant transformants.

4. Selecting such transformants, by appropriate restriction patterns, that have the gpI2O fragment cloned in the proper orientation to generate a fusion protein. When the strain harboring this recombinant plasmid is grown in 2% medium containing 50 sgml ampicillin and the total complement of cellular proteins are electropho'esed on a SDS-polyacrylamide gel, a protein of approximately 12 Kd can be visualized by either coomassie blue staining or by Western blot analysis using as probe selected sera from HIV infected individuals.

IYQnnIP A Purification of recombinant nrotein mntainin~ WV enveinne ~ennences from nlasmid Purification of reco binant nrotein containino HIV envelone senuences from nlasmid pPBI-Sub 1 1. Growth of cells: Cells were grown in a 10-liter volume in a Chemap (Chemapec, Woodbury, NY) fermentor in 2% medium yeast extract, bacto-tryptone, casamino acids [Difco, Detroit, MI], 0.2% potassium monobasic, 0.2% potassium dibasic, and 0.2% sodium dibasic). Fermentation temperature was 30 0 C, the pH was 6.8, and air was provided at 1 win. Plasmid selection was provided 20 yg/mI chloramphenicol.

Typical cell yield (wet weight) was 30 g/l.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-CI pH 5 mM potassium ethylenediaminetetraacetic acid (KEDTA), 5 mM dithiothreitol (DTI), 15 mM )P-mercaptoethanol, 0.5% TRITONTMX-100 (Pharmacia, Piscataway, NJ), and 5 mM phenylmethylsulfonyl fluoride (PMSF). The suspension was incubated for 30 min at room temperature.

This material was lysed using a BEAD-BEATERTM (Biospec Products, Bartlesville, OK) containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1-mmn intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-CI pH 8.0, 5 mM DTT, 15 mM P-ine~cptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized 'using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

I

WO 90/03984 PCT/US89/04302 3. CM Chromatography: The dialysate was loaded onto a 100 ml column (2.5 cm x cm) packed with CM FAST FLOW SEPHAROSE M (Pharmacia) equilibrated in 8 M urea, 10 mM 4-(2-hydroxyethyl-l-piperazine ethane-sulfonic acid (HEPES) pH mM P-mercaptoethanol, and 1 mM KEDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCI. The HIV protein (12 Kd) eluted at approximately 0.2 M NaCI as assayed by SDS-polyacrylamide gel electrophoresis.

Further purification was obtained by pooling Sub 1-containing fractions and applying to a S-200 (Pharmacia) gel filtration column equilibrated in the same buffer as the previous column.

Example 5 Construction of and expression from plasmid pPB1-Sub 2 Plasmid pPB1-Sub 2, which contains approximately 320 bp of DNA encoding essentially the HIV env gene from the Pvull site to the Scal site, and from which is synthesized an approximately 18 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows: 1. Restricting the pPBmIIIB plasmid with Mlul and Seal and isolating the approximately 320 bp fragment.

2. Restricting plasmid pREV2.1 with Mlul and Smal and isolating the large fragment, approximately 4 Kd, from an agarose gel.

3. Ligating the fragment prepared in 2. with the pREV2.1 fragment in a volume of pl using T4 DNA ligase, transforming the ligation mixture into competent cell strain SG20251 and selecting ampicillin-resistant transformants.

4. Selecting such transformants, by appropriate restriction patterns, that have the fragment cloned in the proper orientation to generate a fusion prot eit. When the strain harboring this recombinant plasmid is grown in 2% medium containing 501/g/ml ampicillin and the total complement of cellular proteins electrophoresed on an SDSpolyacrylamide gel, a protein of approximately 18 Kd can be visualized by either coomassie blue staining or by Western blot analysis using as probe selected sera from HIV infected individuals.

Example 6 Purification of recombinant protein containing HTV envelope sequences from plasmid pPBl-Sub 2 L Gro-wth of cells: Cells were grown in a 10-liter volume in a Chemap fermentor in 2% medium. Fermentation temperature was 37 0 C, the pH was 6.8, and air was provided WO 90/03984 PCUS89/04302 31 at 1 wm. Plasmid selection was provided by 20 ug/ml chloramphenicol. Typical cell yield (wet weight) was 30 g/l.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-CI pH 8.0, 5 mM KEDTA, 5 mM DTT, 15 mM Pf.mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. The suspension incubated for 30 min at room temperature.

This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 6 M guanidine-hydrochloride, 20 mM Tris-CI pH 8.0, 5 mM DTI, 15 mM P-mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

The supernate (90 ml) was dialyzed against 4 liters of 8 M urea, 20 mM sodium formate, pH 4.0, 1 mM EDTA, and 15 mM P-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing McGraw Park, IL) with a 3.5 Kd MW cut-off was used.

3. CM Chromatography: The dialysate was loaded onto a 100 ml column (2.5 cm x cm) packed with CM FAST FLOW SEPHAROSE T (Pharmacia) equilibrated in 8 M urea, 20 mM sodium formate pH 4.0, 15 mM P-mercaptoethanol, and 1 mM Na EDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCI. The HIV protein (18 Kd) eluted at approximately 0.2 M NaCI as assayed by SDSpolyacrylamide gel electrophoresis.

Further purification was obtained by pooling Sub 2-containing fractions and applying to an S-200 (Pharmacia) gel filtration column equilibrated in the same buffer as the previous columin.

Example 7 Synthetic peptides Synthesis of peptides can be done by a variety of established procedures, for example, automated peptide synthesis. Peptides were assembled by solid-phase synthesis on cross-linked polystyrene beads starting from the carboxyl terminus and adding amino acids in a step-wise fashion (Merrifield, R.B. [1963] S. Am. Chem. Soc. 85:2149). Each synthesis was pe'formed on an automated peptide synthesizer (Applied Biosystems 430-A) using standard t-Boc chemistry. Amino acids were coupled as highly reactive symmetric anhydrides formed immediately prior to use. To minimize WO 90/03984 Pcr/us89/043022 32 coupling difficulties, dimethylformamide was used as the coupling buffer. The quantitative ninhydrin assay was used to measure the efficiency of coupling after each amino acid addition (Sarin, V.K., S.B.H. Kent, J.P. Tam, R.B. Merrifield [19811 Anal. Biochem. 117:147 1981).

All peptides were deprotected and cleaved from the polystyrene support using an alternative to HF cleavage. Resin containing peptide was resuspended in a mixture of trifluoroacetic acid, trifluoromethane sulfonic acid, and organic thiol scavengers (Tam, W.F. Heath, R.B. Merrifield [1986] J. Am. Chem. Soc. 108:5242). Soluble peptide was precipitated with ethyl ether and, after removing etiher, resuspended in 200 mM sodium carbonate, 3 M guanidine HCI. The crude peptides were purified by reverse-phase chromatography on a 1.0 cm x 25 cm Vidac semi-preparative C1g column. The buffers employed were: 0.1% trifluoroacetic acid in H20, and 0.1% trifluoroacetic acid in 80% acetonitrile/20% HzO. Gradient elution was utilized to elute the bound peptide and collected fractions were further analyzed to identify pure product. Peptide identity was confirmed by amino acid analysis following 6 N HCI hydrolysis. The synthesis included the addition of terminal amino acids not homologous to HIV for purposes of labeling, cross-linking, or structure of the peptide. These non-HIV amino acids are indicated in parenthesis.

The product of synthesis can be further purified by a number of established separatory techniques, for example, ion exchange chromatography.

Example 8 Construction of and expression from plasmid pPBRF Plasmid pPBIRF, which contains approximately 565 bp of DNA encoding essentially the HIVRF env gene from the PvulI site to the BglI site, and from which is synthesized an approximately 27 Kd fusion protein containing this portion of the gpl20 envelope protein can be constructed as follows: 1. Synthesizing DNA fragment in Table 4A.

2. Restricting plasmid pREV2.2 with EcoRV and BamHI and isolating the large fragment, approximately 4 Kd, from an agarose gel.

3. Ligating the fragment prepared in 1. with the pREV2.2 fragment in a volume of pil using T4 DNA ligase, transforming the ligation mixture into competent cell strain CAG 629, and selecting ampicillin-resistant transformants.

4. Selecting such transformants, by appropriate restriction patterns, that have the fragment cloned in the proper orientation to generate a fusion protein. When the strain harboring this recombinant plasmid is grown at 32 0 C in 2% medium containing pg/ml ampicillin and the total complement of cellular proteins electrophoresed on an SDS-polyacrylamide gel, a protein of approximately 27 Kd can be visualized by WO 90/03984 PCT1/US89/04302 33 either coomassie blue staining or by Western blot analysis using as probe selected sera from HIV infected individuals.

Purification of recombinant protein contaiinin HIV envelone sequences frora plasmid Example

PBIRF

e 9

LM

1. Growth of cells: Cells were grown in a 10-liter volume in a Chemap fermentor in 2% medium. Fermentation temperature was 30 0 C, the pH was 6.8, and air was provided at 1 vvm. Plasmid selection was provided by 20 ug/ml chloramphenicol. Typical cell yield (wet weight) was 30 g/l.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 5 mM KEDTA, 5 mM DDT, 15 mM P-mercaptoethanol, 0.5% TRITONTX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for min at room temperature.

This material was lysed using a BEAD-BEATER TM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,006 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-CI pH 8.0, 5 mM DTT, 15 mM Pmercaptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

The supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA, and 15 mM i-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectrophor dialysis tubing with a 3.5 Kd MW cut-off was used.

3. CM Chromatography: The dialysate was loaded onto a 100 ml column (2.5 cm x cm) packed with CM FAST FLOW SEPHAROSE T M equilibrated in 8 M urea, mM HEPES pH 6.5, 15 mM P-mercaptoethanol, and 1 mM Na EDTA at room temperature. The column was washed with 200 ml equilibrium buffer, sad the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCl. The HIV protein (26 Kd) eluted at approximately 0.2 M NaCI as assayed by SDS-polyacrylamide gel electrophoresis.

Further purification was obtained by pooling PBlRF-containing fractions and applying to an S-300 gel filtration column equilibrated in the same buffer as the previous column.

WO 90/03984 PC/US9/04302 34 Example 10 Construction of and expression from plasmid pPBlIN Plasmid pPBlN, which contains approximately 600 bp of DNA encoding essentially the HIVMN eny gene from the BgII site to the BglII site, and from which is synthesized an approximately 28 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows: 1. Synthesizing DNA fragment in Table 2. Restricting plasmid pREV2.2 with BamHI.

3. Ligating the fragment prepared in 1. with the pREV2.2 fragment in a volume of pl using T4 DNA ligase, transforming the ligation mixture into competent cell strain CAG 629, and selecting ampicillin-resistant transformants.

4. Selecting such transformants, by appropriate restriction patterns, that have the fragment cloned in the proper orientation to generate a fusion protein. When the strain harboring this recombinant plasmid is grown at 32 0 C in 2% medium containing 50 pg/ml ampicillin and the total complement of cellular proteins electrophoresed on an SDS-polyacrylamide gel, a protein of approximately 28 Kd can be visualized by either coomassie blue staining or by Western blot analysis using as probe selected sera from HIV infected individuals.

Example 11 Purification of recombinant protein containing HIV envelope sequences from plasmid PPBI N 1. Growth of cells: Cells ware grown in a 10-liter volume in a Chemap fermentor in 2% medium. Fermentaiuot temperature was 30 0 C, the pH was 6.8, and air was provided at 1 wm. Plasmid select;n was provided by 20 pg/ml chloramphenicol. Typical cell yield (wet weight) was 30 g/1.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-CI pH 5 mM KEDTA, 5 mM DIT, 15 mM P-mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for 30 min at room temperature.

This material was lysed using a BEAD-BEATER M containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M u'ret, 20 mM Tris-CI pH 8.0, 5 mM DTT, 15 raM f- WO 90/03984 PCT/US89/04302 mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

The supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA, and 15 mM f-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing with a 3.5 Kd MW cut-off was used.

3. CM Chromatography: The dialysate was loaded onto a 100 ml column (2.5 cm x cm) packed with CM FAST FLOW SEPHAROSE7 t equilibrated in 8 M urea, mM HEPES pH 6.5, 15 mM P-mercaptoethanol, and 1 mM KEDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCI. The HIV protein (28 Kd) eluted at approximately 0.2 M NaCI as assayed by SDS-polyacrylamide gel electrophoresis.

Further purification was obtained by pooling PBlMN-containing fractions and applying to an S-300 gel filtration column equilibrated in the same buffer as the previous column.

Example 12 Construction of and expression from plasmid pPB 1 sc Plasmid pPBlsc, which contains approximately 570 bp of DNA encoding essentially the HIVsc env gene from the PvuII site to the BglII site, and from which is synthesized an approximately 26 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed Vs fallo!s: 1. Synthesizing DNA fragment in Table 6A.

2. Restricting plasmid pREV2.2 with EcoRV and BamHI and isolating the large fragment, approximately 4 Kd, from the agarose gel.

3. Ligating the fragment prepared in 1. with the pREV2.2 fragment in a volume of ,ul using T4 DNA ligase, transforming the ligation mixture into competent cell strain CAG 629, and selecting ampicillin-resistant transformants.

4. Selecting such transformants, by appropriate restriction patterns, that have the fragment cloned in the proper orientation to generate a fusion protein. When the strain harboring this recombinant plasmid is grown at 32 0 C in 2% medium containing g/ml ampicillin and the total complement of cellular proteins electrophoresed on an SDS-polyacrylamide gel, a protein of approximately 26 Kd can be visualized by either coomassie blue staining or by Western blot analysis using as probe selected sera from HIV infected individuals.

WO 90/03984 PCT/US89/04302 36 Example 13 Purification of recombinant protein containing HIV envelope sequences from plasmid pPBlsc 1. Growth of cells: Cells were grown in a 10-liter volume in a Chemap fermentor in 2% medium. Fermentation temperature was 300C, the pH was 6.8, and air was provided at 1 wm. Plasmid selection was provided by 20 pg/ml chloramphenicol.. Typical cell yield (wet weight) was 30 g/l.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 5 mM KEDTA, 5 mM DTT, 15 mM 3-mercaptoethanol, 0.5% TRITONTX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for min at room temperature.

This material was lysed using a BEAD-BEATER T M containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,U00 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-Cl pH 8.0, 5 mM DTT, 15 mM fmercaptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

The supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA, and 15 mM f-mercaptoethanol and 1 mM KEDTA at room temperature. The dialysate was loaded onto a 100 ml column (2.5 cm x cm) packed with CM FAST FLOW SEPHAROSE M equilibrated in 8 M urea, mM HEPES pH 6.5, 15 mM B-mercaptoethanol, and 1 mM KEDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCI. The HIV protein (26 Kd) eluted at approximately 0.2 M NaCI as assayed by SDS-polyacrylamide gel electrophoresis.

Further purification was obtained by pooling PBIsc-containing fractions and applying to an S-300 gel filtration column equilibrated in the same buffer as the previous column.

Example 14 Construction of and expression from plasmid pPB1wMj, Plasmid pPBIWh 2 which contains approximately 560 bp of DNA encoding essentially the HIVwM3 2 env gene and from which is synthesized an approximately 26 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows: WO 90/03984 PCT/US89/04302 37 1. Synthesizing DNA fragment in Table 7A.

2. Restricting plasmid pREV2.2 with EcoRV and BamHI and isolating the large fragment, approximately 4 Kd, from an agarose gel.

3. Ligating the fragment prepared in 1. with the pREV2.2 fragment in a volume of l using T4 DNA ligase, transforming the ligation mixture into competent cell strain CAG 629, and selecting ampicillin-resistant transformants.

4. Selecting such transforma4\ts, by appropriate restriction patterns, that have the gp120 fragment cloned in the proper orientation to generate a fusion protein. When the strain harboring this recombinant plasmid is grown at 32 0 C in 2% medium containing pg/ml ampicillin and the total complement of cellular proteins electrophoresed on an SDS-polyacrylamide gel, a protein of approximately 26 Kd can be visualized by either coomassie blue staining or by Western blot analysis using as probe selected sera from HIV infected individuals.

Exampl pPBl1 e 15 AJ2 Purification of recombinant protein containing HIV envelope sequences from plasmid 1. Growth of cells: Cells were grown in a 10-liter volume in a Chemap fermentor in 2% medium. Fermentation temperature was 30°C, the pH was 6.8, and air was provided at 1 wm. Plasmid selection was provided by 20 pg/ml chlore iphenicol. Typical cell yield (wet weight) was 30 g/l.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-Cl pH 5 mM KEDTA, 5 mM DTT, 15 mM f-mercaptoethanol, 0.5% TRITONTMX- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for 71 iniin at room temperature.

This material was lysed using a BEAD-BEATERTM containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 8 M urea, 20 mM Tris-CI pH 8.0, 5 mM DTT, 15 mM Pmercaptoethanol, 5 mM PMSF, and 1 mM KEDTA. The pellet was solubilized using a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

The supcrnate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM HEPES, pH 6.5, 1 mM EDTA, and 15 mM /-mercaptoethanol. Dialysis was done WO 90/03984 PC-f/US89/04302 38 each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing with a 3.5 Kd MW cut-off was used.

3. CM Chromatography: The dialysate was loaded onto a 100 ml column (2.5 cm x cm) packed with CM FAST FLOW SEPHAROSE T M equilibrated in 8 M urea, mM HEPES pH 6.5, 15 mM i-mercaptoethanol, and 1 mM Na EDTA at room temperature. The column was washed with 200 ml equilibration buffer, and the protein eluted with a 1.0 liter linear gradient from 0-0.4 M NaCI. The HIV protein (26 Kd) eluted at approximately 0.2 M NaCI as assayed by SDS-polyacrylamide gel electrophoresis.

Further purification was obtained by pooling PBlwa2-containing fractions and applying to an S-300 gel filtration column equilibrated in the same buffer as the previous column.

Example 16 Cell fusion inhibition The in vitro fusion of HIV infected cells with T4 positive T cells is measured in the presence and absence of immune serum. This is a well known assay (Putney et al. (1986] Science 234:1392- 1395).

Chronically infected cells and uninfected cells (1:15) are mixed and incubated 24 hr. Foci of fused cells (giant cells) are then counted (usually about 60). Dilution of an immune serum, for example, serum to the entire HIV envelope (gpl60) or to a protein or peptide of the invention, is added when the cells are mixed. A 90% decrease in giant cells after 24 hr indicates the immune serum can block fusion. This assay can be done with cells infected with various virus strains, for example, HIV B and HIVRF.

Example 17 Competition cell fusion Using the assay described in Example 16, one can determine if proteins or peptides contain the epitope recognized by antibodies that are responsible for cell fusion inhibition. For example, fusion inhibition of HIVIIIB infected cells by antiserum to the PB1-IIB protein of the parent application is abated by addition of PBI-III protein to 5 /g/ml. Using antiserum to PBI-11 1 and adding any one of the proteins or peptides, for example, Sub 2, Sub 1, CNBrl, peptide 135 or peptide 136 at 5 pg/ml totally blocks the activity of the PB1 antiserum. Additionally, antiserum to PBl-RF that is capable of neutralizing HIV-RF can be blocked in this activity by peptide 139. A peptide containing only the central portion of the peptide 139, peptide 339, also can block the fusion inhibition activity of antiserum to PB1-RF. This, for the first time, localizes the critical amino acids WO 90/03984 PCT/US89/04302 39 necessary to elicit neutralization or block fusion inhibiting antibody to a ten amino acid sequence peptide 339).

Example 18 Co-immunization of

P

B1-IIIg and PB1RF Antisera from an animal immunized with two PB1 proteins from HIVln and HIVRF isolates were capable of blocking cell fusion of both HIVIja- and HIVRF-infected cells. This d imonstrates that co-immunization with separate proteins containing envelope sequences of two H'V isoi't_-s elicits an immune response capable of neutralizing both isolates. This novel property of small proteins or peptides blocking immune seniru has not bee:? described before.

Some of the proteins and peptides of the subject invention contain the entire epitope for raising humoral immune responses that neutralize HIV infection and block HIV infected cell fusion.

This is shown by these proteins and peptides competing these activities out of serum from animals immunized with the entire HIV envelope. More specifically, proteins and peptides that can compete the activities from anti-gp360 or anti-PB1 sera are Sub 2, Sub 1, CNBrI, peptide 135, and peptide 136.

The proteins and peptides of the invention also can be used to stimulate a lymphocyte proliferative response in HIV infected humans. This then would stimulate the immune system to respond to HiV in such individuals.

Example 19 Construction of and expression from plasmid pPBllig Plasmid pPB1, which con.ains approximately 540 bp of DNA encoding essentially the HIV env gene from the Pvull site to the BelII site, and from which is synthesized an approximately 26 Kd fusion protein containing this portion of the gpl20 envelope protein, can be constructed as follows: 1. Synthesizing the DNA with the sequence shown in Table 8: This DNA fragment can be synthesized by standard methods and encodes a portion of gpl20. It has a blunt end on the 5' end anO an end which will ligate with a BamHI overhang on the 3' end.

2. Restrictini: 5 pg plasmid pREV2.2 with EcoRV and BamHI and isolating the large fragment, approximately 4 Kd, from an agarose gel.

3. Ligating 0.1 pg of the fragment in Table 8 with 0.1 pg of the pREV2.2 fragment in a volume of 20 I1 using T4 DNA ligase, transforming the ligation mixture into competent cell strain SG20251, and selecting ampicillin-resistant transformants.

Using the Ahall restriction pattern of purified plasmid, selecting cells harboring the recombinant plasmid with the synthesized fragment in he orientation whereby the fragment blunt end ligated to the REV2.2 EcoRV end and the BamHI overhanging ends ligated together. AhalIl digestion of the proper plasmid gives fragment lengths of approximately 1210, 1020, 750, 690, 500, 340, and 20 base pairs. When the strain WO 90/03984 PCT/US89/04302 harboring this recombinant plasmid is grown in 2% medium containing 50 plg/ml ampicillin and the total complement of cellular proteins electrophoresed on an SDSpolyacrylamide gel, a protein of approximately 26 Kd can be visualized by either coomassie blue staining or by Western blot analysis using as probe selected sera from AIDS, ARC, or HIV infected individuals.

Example 20 Purification of recombinant protein containing HIV envelope sequences from plasmid nPBIIIB 1. Growth of cells: Cells were grown in a 10-liter volume in a Chemap fermentor in 2% medium. Fermentation temperature was 37 0 C, the pH was 6.C, and air was provided at 1 wm. Plasmid selection was provided by 50 pg/mi ampicillin and 20 pg/ml chloramphenicol. Typical cell yield (wet weight) was 30 g/l.

2. Cell lysis: 50 g, wet cell weight, of E. coli containing the recombinant HIV envelope fusion protein were resuspended in a final volume of 100 ml in 50 mM Tris-CI pH 8.0, 5 mM KEDTA, 5 mM DT, 15 mM f-mercaptoethanol, 0.5% TRITON' X- 100, and 5 mM PMSF. 300 mg lysozyme was added and the suspension incubated for min at room temperature.

This material was lysed using a BEAD-BEATER

T

M (Biospec Products, Bartlesville, OK) containing an equal volume of 0.1-0.15 mm glass beads. The lysis was done for 6 min at room temperature in 1 min intervals. The liquid was separated from the beads and centrifuged for 2.5 hr at 20,000 xg. The supernatant was removed and the pellet was resuspended in 100 ml 6 M guanidine-hydrochloride, 20 mM Tris- Cl pH 8.0, 5 mM DTT, 15 mM B-mercaptoethanol, 5 mM PMSF, and 1 mM KEDTA.

The pellet was solubilized usisg a polytron homogenizer and centrifuged at 20,000 xg for 2 hr.

The supernate (90 ml) was dialysed against 4 liters of 8 M urea, 20 mM Tris- Cl, pH 8.0, 1 mM EDTA, and 15 mM 6-mercaptoethanol. Dialysis was done each time for 8 hr or longer with three changes of buffer. Spectraphor dialysis tubing (S/P, McGraw Park, IL) with a 3.5 Kd MW cut-off was used.

3. CM Chromatography: The dialysate was loaded onto a 550 ml column (5 cm x 28 cm) packed with CM FAST FLOW SEPHAROSE TM equilibrated in 8 M urea, mM potassium phosphate pH 7.0, 15 mM P-mercaptoethanol, and 1 mM KEDTA at room temperature. The column was washed with 2 liters equilibration buffer, and the protein eluted with a 5.0 liter linear gradient from 0-0.4 M NaCI. The HIV WO 90/63984 PCr/US89/04302 41 protein (26 Kd) eluted at approximately 0.2 M NaCI as assayed by SDS-polyacrylamide gel electrophoresis.

Example 21 Immunization with two or more peptides to obtain broadly neutralizing antisera Five peptides, peptide 135, peptide 139, peptide 141, peptide 142 and peptide 143, were cross-linke4' individually to carrier proteins and used to immunize goats. Each peptide is capable of eliciting tye specific neutralization when used individually as an immunogen. Synthetic peptides were crois-linkd through a sulfhydryl bond to keyhole limpet hemocyanin (KLH) by using Succinimidyl- 4-(n-Maleimidomethyl)Cyclohexane 1-Carboxylate (Pierce). The ratio of peptide to KLH was 1:2 by weight. 200 pg of each cross-linked peptide was used in the immunization cocktail (a total of 1 mg of 5 peptides, 2 mg of KLH). This method of crosslinking or immunization regimen is but an example and not meant to be limiting. After four immunizations, immune sera wa: ested for neutralization of these five HIV isolates as well as distinctly different isolates. The immune serum could block fusion of cells infected with any of five isolates from which the peptide sequences were derived. In addition, the serum neutralized other variants not used in the immunization.

Equivalent broad neutralizing sera may also be obtained by variations of this immunogen. For example, using more than five peptides having the amino acid sequence derived from the principal neutralizing domain from more than five variants. Alternatively, a single peptide peptide 64' or peptide 74) containing segments homologous to diverse HIV variants may also be used to elicit broad neutralizing antibody.

Example 22 Sequential Immunization with Two or More Peptides as a Method to Elicit Broad Neutralizing Antisera An immunization protocol capable of eliciting broad neutralizing antibodies may take the form of initial immunization with a peptide or protein antigenically equivalent to the principal neutralizing domain, or segments thereof The initial immunization is followed with a second immunization. The initial immunization could be done with, for example, peptide 135, peptide 139, peptide 141, pept'de 142, or peptide 143, with subsequent immunization with, for example, one or more of the following peptides: RP57 Ile Asn Cys Thr Arg Pro Ala His Cys Asn Ile Sr Ala His Cys Asn le Ser (Ala Ala Ala Ala Ala Ala) Gly Pro Gly Arg (Ala Ala Ala Ala Ala Ala Cys) RP56 Ile Asn Cys Thr Arg Pro RP59 lie Gly Asp lie Arg Gin Ala His Cys Asn lie Sr WO 90/03984 PC/US9/04302 42 The method is to immunize with a protein or peptide and then boost the immune response to a defined subset of the original immunogen. This immunization method may be useful in vaccine methodology and also to generate broad neutralizing polyclonal or monoclonal antibodies ,or therapeutic applications.

Example 23 Identification of Critical Segments of the Principal Neutralizing Domain Certain segments of the principal neutralizing domain have been found to be capable of eliciting the antigenic and immunogenic responses which are associated with the principal neutralizing domain as a whole. For example, a region of the principal neutralizing domain known as the "tip of the loop" has been shown to be capable of raising, and/or binding with, neutralizing antibodies. This capability is observed for the "tip of the loop" of a variety of HIV variants.

The tip of the loop comprises a three amino acid segment which is highly conserved between HIV variants, together with various amino acids which occur on either side of the three conserved amino acids. The three conserved amino acids, which are Gly Pro Gly, usually occur at, or about, positions 311, 312, and 313 of the HIV envelope protein.

The "tip of the loop' comprises the Gly Pro Gly segment together with the 2 to 8 amino acids which flank either or both sides of this segment in any given HIV variant. The amino acids which flank the conserved segment may be any of the 20 natural amino acids, in any sequential order.

Although the amino acid sequence of the principal neutralizing domain varies between different HIV- 1 isolates, conservation at particular positions, for example at the tip of the loop, suggests that certain amino acids at these positions are necessary for virus function.

Example 24 Sequence of the Principal Neutralizing Determinant from Randomly Selected HIV-1 Isolates Sequences of the principal neutralizing domains (PNDs) from random field isolates were obtained in order to determine the degree of heterogeneity within this region of the envelope protein.

Peripheral blood lymphocytes (PBLs) from randomly selected HIV-1 infected donors were either cocultured with uninfected PBLs or the virus isolates were adapted to CD4 cell lines. DNA was extracted from these infected cells and a 240 base pair region encoding the PND was amplified by polymerase chain reaction using oligonucleotide primers that hybridize with flanking conserved regions.

This product was cloned into pUC19 and the sequence of the PND from one or more clones from each original isol :te were determined. Because of the heterogeneity of the virus population within one infected individual, when two or more sequences were obtained from one PCR reaction, these sequences sometimes differed.

WO 90/03984 PCr/US89/04302 43 The data obtained from nearly 100 individuals (some infected with a heterogeneous virus population) was evaluated along with previously obtained HIV sequence information. Table 9 lists 138 PND sequences from HIV isolates. These sequences indicate that, despite the well-known and frequently cited variability in the amino acid sequence of the HIV envelope protein, there is actually a high degree of conservation in the immunologically critical PND region, particularly in the region at the center of the PND. Specifically, the Gly-Pro-Gly sequence at the "tip of the loop" occurs in over 90% of the variants. Furthermore, other amino acids at certain positions on each side of the G- P-G were also found to be highly conserved. Table 10 shows the frequency of occurrence of the various amino acids at each position in the PND. In addition to the very strong conservation of the glycines flanking the central proline, there is strong conservation at several other positions R at

X

12 P at x 1 l, G at yil, R at y 1 4 and A at y 16 A comparison of the relative frequency of variations of a 17-amino acid segment centered about the G-P-G sequence is shown in Table 11. In this table, the sequence which reflects the most commonly occurring amino acids at each position is listed first. The dashes indicate identity with the consensus sequence. The remaining sequences are ordered from 2 to 138, according to their homology to the consensus sequence. Thus, the sequences at the top of the table display the greatest homology with the consensus sequence. Sequences far down the table display less homology. For example, the amino acid -equences from isolates IIIB and LAV-BRU occur at positions 92 and 93, respectively, on this table. This indicates that these isolates have only limited homology with the consensus sequence.

The present research shows that HIV viruses such as IIIB and LAV-BRU having the Gin-Arg (Q-R) dipeptide to the left of the Gly-Pro-Gly sequence are relatively uncommon. By contrast, the MNlike sequence in this region H I G P is the most common. The present research shows that principal neutralizing domains of other commonly studied variants comprise relatively uncommon sequences.

Although the subject invention pertains to the discovery of certain highly conserved regions in the principal neutralizing domain, there remains some degree of variability in this region among the various isolates. This variability includes "missing" or "added" amino acids at certain points in the sequence. Of course, "missing" or "added" amino acids can cause difficulty in devising aesthetically pleasing tables showing the sequences. However, these missing or added amino acids pose no difficulty to a person skilled in the art in terms of locating the highly conserved regions which are critical to the subject invention. Table 9 shows one representation of 138 PND sequences. Table 11 uses a slightly different representation to show the same PND sequences. The primary discussion of sequence conservation can probably best be visualized by reference to the representation shown in Table 11.

However, it should be noted that the existence of more than one means for representing these

IF

A I II WO 90/03984 PCT/US89/04302 44 sequences does not compromise the ability of the skilled artisan to accurately locate the sequences or the conserved regions.

With the discovery of commonly occurring amino acid sequences, it is possible, for the first time, to develop prophylactic and therapeutic compositions which can predictably elicit and/or bind with neutralizing antibodies to a broad range of HIV variants. The generalized formula for such a composition can be as follows: axGzGyb wherein x is 0 to 13 amino acids i length; y is 0 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T-cells, or a general immune stimulant Further analysis of common sequence patterns reveals certain specific patterns which are very common among HIV isolates. Examples of these common sequences are shown in Table 12 and Figure 1. These common patterns, which are known only as a result of the research and discoveries described here, can be used to make pharmaceutical and diagnostic compositions which can be used with a broad range of HIV isolates. This, of course, can be of critical importance, given the large number of HIV variants which are now known to exist Table 12 is a compilation of the common sequence patterns that occur in the region at the tip of the loop. For example, approximately 60% of the HIV isolates contain the core sequence I a I G P G R (a represents several different residues), approximately 50% contain the sequence I G P G R A, and approximately 40% contain G P G R A F. When a His residue is present at the a position in I a I G P G R, this sequence occurs in approximately 30% of the HIV isolates. A vaccine composition comprising a mixture of peptides having the sequence I a I G P G R where all of the possible replacements for the a are present, is capable of eliciting antibodies which neutralizes a majority of HIV variants. Preferably, for use as immunogens, the peptides are linked to carrier proteins or adjuvants as described in Example 21.

As shown in Figure 1, common sequence patterns are also apparent within the 17 amino acid segment. Sequences which were isolated 4 or more times are highlighted. These commonly occurring sequences can be used to formulate vaccine cocktails which elicit a broadly neutralizing response. For example, a potential cocktail might contain peptides from each of the eight groups represented.

Alternatively, the peptide sequences may be presented as a hybrid polypeptide containing the principal WO 90/03984 S90 0 PCr/US89/04302 neutralizing domain from two or more of these groups. Preferably,, such a cocktail will contain peptides which will be capable of raising antibodies which neutralize at least 70% and most preferably at least 90% of HIV variants.

The antigens of the subject invention can be identified by their ability to raise antibodies which bind to certain amino acid sequences. For example, particularly advantageous antigenic compounds would raise antibodies which bind to common amino acid sequences such as G-P-G-R- A-F, I-G-P-G-R-A-F, I-a-I-G-P-G-R, I-a-1-G-P-G-R-A, and l-a-I-G-P-G-R-A-F, where a is any of the 20 amino acids.

From Table 10 it can be seen that a polypeptide representing the occurrence of amino acids in all of the variants can be represented as follows: X13 X12X1 IxO X9 X8X7 X65x5 4 x 3 x 2 xG zG Y 2

Y

3

Y

4

Y

5

Y

6

Y

7

Y

8

Y

9

Y

10

Y

11

Y

12

Y

13

Y

14

Y

5

Y

16

Y

17 wherein X is1, R, M,lQR, V,1, K, F,S,0, Y,SRG, orYQR; x 2 is H, R, Y, T, S, P, F, N, A, K, G, or V;,

X

3 is 1, L, M, T1, V, E, G, F, or Y;,

X

4 is R, S, 0, H, A, K, or not present; x 5 is K, R, 1, N, Q, A, IR, RQ, or not present;,

X

6 is R, P, Q, E G,orT;

X

7 Is T, K, V,I1, A, R, P, or E;

X

8 is N, NV, Y, 1, T, DK, H, or x 9 is N, S, E, Y, D, 1, or Q; x1O is N, Y, So G, or H; x 11 is P;

X

12 is R, 1, or Y4

X

13 is T, 1, M or A; z i~s P, A, 0, S, or 14 Yis R, K, 0, G, S, or T;, y2 isXV, N, R,K, T, S, F,P,or W; Yis F, 1, V, L, W, Y, G, 5, or T; 314 Is Y, V, H, I, F, S, 1, T, M, R, X'H, or Fl'; is T, A, V, 0, H, 1, S, Y, or not present; Y16 is T, R, 1, Q, A, M, or not presenv WO 90/03984 PCT/US89/04302 46

Y

7 is G, E, K, R, T, D, Q, A, H, N, P, or not present; yg is R, Q, E, K, D, N, A, G, S, I, or not present; y 9 is I, V, R, N, G, or not present; Yio is I, T, V, K, M, R, L, S, E, Q, A, or not present; Yul is G, R, E, K, H, or not present; Y12 is D, N, I, R, T, S, or not present; Y13 is I, M, ME, L, or not present;

Y

14 is R, G, K, S, E, or not present; yis is Q, K, or R; y 1 6 is A; and Y17 is H, Y, R, or Q.

Monoclonal and/or polyclonal antibodies with broad neutralizing activity can be generated using the commonly occurring peptide sequences for use in prophylactic or therapeutic compositions.

The commonly occurring sequences described here can be used in much the same way as the other peptides described in this application. For example, these peptides can be modified in order to provide T-lymphocyte stimulation, general immune stimulation, to enhance immunogenicity or solubility, or to reduce toxicity. The peptides may also be modified by addition of terminal cysteine residue(s) or by conjugation to a carrier protein, adjuvant, spacer, and/or linker. The peptides may be fused with other HIV epitopes to produce a multiepitope polypeptide which could be useful with an even greater number of HIV variants. Also, the peptides can be circularized by bonding between cysteine residues. The cysteine residues used to make such circularized peptides could be the naturally occurring cysteine residues at the ends of the principal neutralizing domain, or cysteine residues may be added to the terminal ends of the peptides.

Additionally, vaccine compositions may include peptides containing T helper cell epitopes in combination with protein fragments containing the principal neutralizing domain. Several of these epitopes have been mapped within the HIV envelope, and these regions have been shown to stimulate proliferation and lymphokine release from lymphocytes. Providing both of these epitopes in a vaccine composition may result in the stimulation of both humoral and" cellular immune responses.

Example 25 Construction and Cloning of Multi-Epitope Genes Synthetic genes can be constructed which encode proteins comprised of the neutralizing epitopes from more than one HIV isolate. The synthetic gene exemplified here comprises a tandem arrangement of DNA sequences encoding neutralizing epitopes from HIV isolates IIIB, RF, SC, MN, and WMJ1. Each epitope-encoding domain within the gene was designed to encode the 11 amino WO 90/03984 PCT/US89/04302 47 acids centered at the common Gly-Pro-Gly sequence at the tip-of-the-loop for each of the isolates.

Thus, the multi-epitope gene contained 5 different coding regions, each of which encoded a neutralizing epitope from a different isolate. For this particular construction, the epitope which was chosen for each of the 5 isolates consisted of the Gly-Pro-Gly sequence along with the 4 amino acids on either side of the Gly-Pro-Gly sequence from each of the 5 isolates. Domains coding for other neutralizing epitopes from these isolates could have been" incorporated into the multi-epitope gene.

Also, genes coding for neutralizing epitopes from other isolates can be used.

The genes were constructed such that the domains were linked by DNA sequences encoding four glycine residues. The composition or length of the linking sequence can be varied but preferably it is a sequence that is non-immunogenic itself. The DNA sequence of the synthetic gene described here was designed such that restriction sites were encoded at either end of the fragment to facilitate cloning into the vector or, alternatively, to permit the construction of longer multi-epitope genes by attachment of 2 or more shorter genes (Figure In addition, a methionine residue was encoded at the 5' end of the gene to facilitate cleavage when produced as part of a fusion protein.

Figure 3 depicts the steps in the construction of the multi-epitope gene described here. The amino acid sequence encoded by this gene is shown in Table 13. The portions of this amino acid sequence which correspond to each of the 5 isolates are identified in Table 13.

Double-stranded subfragments of the full-length gene were first constructed starting with single-stranded synthetic oligomers designed to encode tandem neutralizing epitopes and linking amino acid sequences. Any number of subfragments can be used. In this experiment the gene was divided into two portions, but three, four, or more portions can be used. Four single-stranded oligomers of between 67 and 78 nucleotides in length were synthesized (HEO-1, HEO-2, HEO-3, and HEO-4) (Figure The oligomers were designed in pairs (HEO-1 and 2; HEO-3 and 4) as opposite and adjacent strands of the double-stranded subfragments having 10 (HEO-1 2) or 11 (HEO-3 bases of complementary overlap. The oligomers of each pair were mixed and heated 65'C for 5 minutes, then incubated at 37"C for 1 hour to anneal.

After annealing, the complementary strands of each pair were completed ("filled-in") using Sequenase Biochem) and the four deoxynucleotide triphosphates. This reaction was incubated for 1 hour at room temperature, heated at 65'C for 3 minutes, and then incubated for an additional hour at 37'C with fresh Sequenase. Double-stranded fragments of 141 (HEO-1+2) and 126 (HEO- 3+4) base pairs were generated representing adjacent subfragments of the multi-epitope gene. HEO- 1+2 comprised the coding sequences for 3 epitopes plus adjacent linker amino acids; HEO-3+4 extended from the fourth epitope to the end of the gene.

Following the fill-in reaction, the samples were extracted with phenol/chloroform and precipitated with ethanol by standard procedures. The resulting double-stranded DNAs were digested WO 90/03984 PCT/US89/04302 48 with HindIII (HEO-1+2) or Sac (HEO-3+4) and purified on a 3% NuSieve agarose gel. The purified fragments were ligated with HindIII Sacl digested pUC19 (New England Biolabs) in a 3component ligation and transformed into E. coli JM105 cells. The presence of a 256 base pair fragment in pUC19, encoding the full-length multi-epitope gene, was confirmed by restriction analysis and DNA sequencing. The resulting plasmid was designated pUC/MEP-1.

The MEP-1 insert was removed from pUC/MEP-1 and recloned into HindllI Sacl digested pRev2.1 for high-level expression of a fusion protein comprised of a leader portion from the E. coli BG gene fused to the multi-epitope protein. The resulting plasmid, designated pMEP-1-8342, was transformed into E. coli strain SG20251 and the 12.9 Kd multi-epitope fusion protein was identified by coomassie blue staining or Western blot analysis using a probe selected from antisera to the looptip peptides from each of the 5 HIV isolates. The fusion protein can be used intact or, alternatively, the leader portion can be cleaved off by cyanogen bromide which cleaves on the carboxy-terminal side of methionine residues. The amino acid sequence of the fusion protein is shown in Table 13A.

The multiepitope'peptide can be purified from recombinant cells by methods described above.

Other synthetic genes can be constructed which encode tandem neutralizing epitopes from any number of different HIV isolates using the procedure described above. In addition, variations on the above procedure can be made which are meant to be included in the present invention. For example, the lengths of the neutralizing epitopes encoded by a gene can vary, and there can be variation in the length of the individual epitopes within a single gene. Further, the number of neutralizing epitopes within a multi-epitope gene can vary, and the composition or the length of the amino acid sequences of the epitopes or the linking sequences can be varied from the example that is described herein.

It should be understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes-in light thereof will be suggested to persons skilled in the art and are to be included within the spirit and purview of this application and the scope of the appended claims.

This work was supported in part under contract number N01-AI-62558, awarded to Repligen Corporation by the National Institute of Allergy and Infectious Diseases (NIAID).

=4 1W 143tgp 160 295 474 51a 759 gp 120 gp 41 ill 181110)

RF

MN

Sc WMJ.2

LAV-MAL

SF-2 Z3 WVMJ1 WMJ3.

Z6

LAVELI

CD C451 CD C4 2

BAL

HIV-2 -T R V M HUHT R K 8 1 R! Q R 9 P RA I V T I G R 1 0 H U R 9 A 9 F

SDKKI

D-I-

T K H I-- A T D I -T I v A R I A K- G I T -TRGRT- I -TRSRS I -T -I I L -T I IL- T-Z I-- K- ills (BHIO)

RIF

MN

Sc WMJ-2

LAV-MAL

S F-2 Z3

WMJI

WMJ3

LAVELI

CDC451 CD C4 2

BAL

-I-

-I-

G -V 9- MLH S H V R 8 E Y Q PIN K R I' i-IV-2 Table 1.

WO 90/03984 PCT/US89/04362 TABLE 2 L e u A s ri G I rS e r Va 1 G 1 u I I eA sr nC v sTh r A r9F r oA sAsr As As r T h r A rg~Lv s G I A s rte t A r!G rI A I aH i sCws Asr I Ie Se rA r9A Ia Lvs*r pA As nA s riTh r TABLE '-A CTBA A CCA AT CT GTAGiA A T TA ATT GT ACAA GA C CCAA C AA CAAT ACA AG AA AA AG TAT CCGBTAT CC AGAG AG GA CC AG GG AGA CATTT G TTA C AAT AGGA A A AATA GGBAA ATAT GAG AC AAG C ACAT T GTA A CATTAG TAG AGC AA AAT BGA ATAA C ACTT T TABLE 2B Tr1F-Al1aFhe S erLeuAsF-Ar-G luArgVal Al aAspLeuAsriG1 riSe pVal1Glu1 ileAsriCvsThrAr-qFi-oAsr:AsriAsraThrArgL-dsSerIl1eAprI 1 eG 1rgATrs B 1 'MroB 1 vA r5sA aFhelalITh rileG1 Lvs le G I A sri~etA r-cGI riM p HisCvsAsrih-leSerAnAlaLysTrAsrAsnThrLeuGlvAlaATr IeLt-ii TABLE 2C ATBTTACGTCCTGTABAAAfC'CCAA "CCTGAAA TCAAAAAACTGGACBGCV-TO TB B B CAT7 t A TC TBBAT ICCBA A C BCGBTB BC CBAT CTB A ACC AATC TB TrA AA ATTAA7TTACAAGACCC"AACAACAATACAAGAAAAAGTATCCGTATCCAGAGA B BA C A BBGAGA BC AT TTB T T ACA ATAG BAAA A ATAG BAA ATAT GABA C AAGC A

CATTGTAACATTAGTAGAGCAAAATGGAATAACACTTTBGGABCTCBAATTCTT

B AAG A AGGGiL'ii zCTC WO 90/03984 PC1'/US89/04302 51 TABLE 3 L eui As rj G 1 ri S e rV a 1 6 1 uI le As nCdsT h r A r.9 Pr roAs rnAsn~As riT h r A L s 01 -AsiM e tA r-qGrI A I ali sCvs A sr 1 e S e rArgA I aLw sT rr-A s riAs riTh r 11 eFheL s G I riSe r S e PG 1vG 1 As,:4 ro G 1 u I 10 B I'aTh rH i s Se r Ph eA s r Cvs jG1vG1uh~ ~,Cs n rT hrG I iL e u~ieA srnSe r TABLE 3A CT GA ACC AAT CT GTAG A A AT TA ATTOGT AC AA GA CC CA ALL'A A C A AT A C AA G A A AnA AG TAT C COT AT CC AGAG AG GA CCAG G GA G AG1 C AT TT 6T TAC AAT A G GA P.A A T A GGAAATATGAGACAAGCA CATTOTAACATTAGT AG A GAAAATGGAATA AC AC T T TAA AA C AGA TAG ATA G C AAATTA AAGAG A AC A ATT T GGA AAT A ATA A A AA CA A TA AT CT TTAAG CAG TC CT CA GGAGG66GA CCC AG A A ATT O T AA C GC AC AG T T T T A AT TT 76 GAG 666G A AT TT TT C TACTS6TA AT TC A A C A C A ACT G TTT ATA A G T WO 90/03984 WO 9003984PCT/US89/04302 52 TABLE 3B Me~ur9,r~-IG ~rr~rr ~jcl11de T rr-'Al a F'he Se r e uA sFA r Diu Arg~Vpa Al a As p-L eujA sril 01nS e rV a 1G 1 uj IleAsriCdsTh rA r9F'roAsriAsrAsriThrAr9LvsSe rI 1 eAr!s Ii Gi riArgi Bi yF' r o B yA rg~Al a F'he Va 1 Thrl 1 el yLv~s le I1 e~lAs ri ~e tA r.g ln0 1 ria HisrZvsAsriI 1 e Se rArslAl eLvsT rrpAsriAsriThrLeuLvsGlrtI 1 eAsr- Setr L-,sLeujAr9G 1 uGi ri'he~i vAsriAsr;LvsThr 11ie I I eF'heLdsG 1 riSe rS e r B 1 il yAs pP r o I ul I eVa iT hrHi s Be r Ph eA sriCvysO 1 G 1 v uF he Phe Tvr Cv~sAs ri Sc r Thr B 1rLe uF' he AsnrSe rl 01 yS e r Se rAs riSe r TABLE 3C A TB TTAC BTC CT B TAG AA A CCCC AA CC C GTGA AAT CA AA A A AC TGGA CG G C CT G TB GB CAT TCAB TC TO3G AT COCAA COCTOTBC C BAT CTOA A CC AAT CT GTAG AA ATTAATT GTirAAAOACC-CAACAACAATACAAGAAAAAOTATCCOTAT CCABAGA B BA CC A GBGA GAO AT TT OTT A C AATAOBAAA AAT AGB AA ATATBAGBA C AAGC A CAT TB T AA CF-- T A B T A BA BC A AA ATOGA ATA A CACT TTA A A AC AAT AG IAT AGBC A AAT TA AGAG AA CA ATi GGB AA ATA ATA AA AC A ATAAT CT TTAAG C AGT CCTC A GBAGGACCC,-A GA AATTBT AA CO CACAO TTTTA AT TGT GAGGOAA TT TTT C TACTGBT AA*-T TC IA A AC A AC TOT TTA ATAG TOBGAO CTC BA AT TCT -53 TABLE 4 LeuAsnAlaSerValGlnI 1eT~srCysThrArgProAsnAsrnAsnThrArgLys Ser leThrLysGlyProl~lyArgVal IleTyrAl aThrGlyGlniIl]eI leGly AspI leArgLysAl aH1.sCysAsnLeuSerArgAlaG lnTrpAsnAsnThrLeu LysGlnValValThrLysLeuArgGiuGlnPheAspAsnLysThrl leVaiPhe ThrSerSerSerGlyGlyAspProGlut leVal LeuHisSerPheAsnCysGly GiyGluPhePheTyrCysAsnmhrThrGlnLeuPheAsnSerThrTrpAsnSer ThrGluGlySerAsriAsnThrGlyGlyAsnAspThrI leThrLeuProCysArg I 1eLysGlnhleVa1AsnMetTrpGlnGluValGlyLysAlaMetTyriAlaPro ),roI2.1,eSerGl-yGnhleLysCysIleSerAsnI leThrGlyLeuLeuLeuThr ArgAspGlyGlyG uAspTh. ThrAsnThrThr TABLE 4A CTGAATGCATCTGTACAAATTAATTGTACAAGACCCAACAACAATACAAGAAAAi

GACTTACGTAGACATGTTTAATTAACATGTTCTGGGTTGTTGTTATGTTCTTTT

AGTATAACTAAGGGACCAGGGAGAGTAATTTATGCAACAGGACAAATAATAGGA

TCATATTGATTCCCTGGTCCCTCTCATTAAATACGTTGTCCTGTTTATTATCCT

GATATAAGAAAAGCACATTGTAACCTTAGTA(JAGCACAATGGAATAACACTT TA

CTATATTCTTTTCGTGTAACATTGGAATCATCTCGTGTTACCTTATTGTGAAAT

AAACAGGTAGTTACAAAATTAAGAGAACAATTTGACAATAAAACAYATAGTCTTT

TTTGTCCATCAATGTTTTAATTCTCTTGTTAAACTGTTATTTh'GTTATCAGAAA ACGTCATCCTCAGGAGr~X-GACCCAGAAATTGTACTTCACAGTTTTAATTGTGGA

TGCAGTAGGAGTCCTCCCCTGGGTCTTTAACATGAAGTGTCAAAATTAACACCT

GGGGAATT~TTCTACTGTAATACAACACAAGTGTTTAATAGTACTTGGAATAGT

CCCCTTAAAAAGATGACATTATGTTGTGTTGACAAATTATCATGAACCTTATCA

A\CTGAAGGGTCAAATAACACTGGAGGAAATGACACAATCACA eTC( CATGCAGA TGACTTCCCAGTTTATTGTGACCTCCTTTACTGTGTTAGTGTG"I GGTACGTCT ATAAAACAAATTGTAAACATGTGGCAGGAiAGTAGGAAAAGCAATGTATGCCCCT

TATTTTGTTTAACATTTGTACACCGTCCTTCATCCTTTTCGTTACATACGGGGA

CCCATCAGTGGACAAATTAAATGTATATCAAATATTACAGGGCTACTATTAACA

GGGTAGTCACCTGTTTAATTTACATATAGTnT ATAATGTCCCGATGATAATTGT At3AGATGGGGGTGAAGATACAACTAATACTACAGA TCTCTACCCCCACTTCTATGTTGAi'TATGATGTCTCTAG 921130,p:%oper~eAb44036-89.rsp5 wVo 90/03984 PCT/US89/04302 54 TABLE 4B Met L e u A r8 P F ro a 1. G 1 u T h rF Pr0Th r A r! 6 61 u 1 1 e L tj s L v s L e u A s r, G1 I L e u T rA Al a Ph e S e r L e ui A s F, A r' 0 61 t.UA r! V a 1 A 1 a A s P L e u A sri AI a S e r V) p I G1 ni 11 eAsriC -as Th rA r SFr o A s nAs~AsriTh rA r9Lvs Se r 11 e Th r L ,s G 1. PiFvo 61Y Va11eTwr G1r 1e1 31- -IIeAr, CysAsrLeuSerAr9Al aClriTrpAsrgAsriThrLe'jLy-s61 nVa 1 Val2 Th rLv s Leu A rgG I uGl1rn h e As r-AsriL vsTh r I I e Va 1FPh e Th r So r Se r Se rO G 1,jG 1 'i AsFFroG61 u 11 e Va 1 Le uH i s Se rP he AsiCv sl G 1 .jGIjG1i P~he Ph eT v r C vs As rThrTh rGIrnL e uF' h e As riSerTh TT A sriS e r Th r 01 u, G I vS e rA sriA srn Th rG yG wA s A s Th r 1eTh rL ei Pr oC s Ar !1e Lvs Glr, 1 eV e1 As r MeTr AIaeTurA1aP 1eSer gGIn1 LysCv s 11e Se rA sr n 1 e Th rG jL euL euL e u7h r Ar!-A s P G IYG 1 y G 1 uJA sP Th hrT hr A~ sri T hr Th rG 1 u I I e A PA Ar9 ~1 nA Aa S e r A r 9 G 1 ii L e u G 111 FPh e L e u LqsTh rL es GvPr cA rgA s -Th rFr o 11ePhe I e G WO 90/03984 PCT/ US89/04302 TABLE 4C ATGTTACGTCCTGTAGAAACCCCAACCCGT GAAAT CAAAAAACT GG AC GGC CT G

TGGGCATTCAGTCTGGATCGCGAACGCGTGGCCGATCTGAATGCATCTGTACAA

ATT AATT BT AAGACCCAACAAC-AAT ACAA A A AAA-GTAT A ACTA AGG A C CA G GO GAG AG TAAT TTAT GCA A C AG CA A ATA AT AG GAGA AT A A GA A A A G C A C A T TOGTA AA I TT AG T AGAG CA CA AT GGA ATA A CA CT TT AA A AC AG G TAG OTT A C A A A A TT AA GAGAACAATTTGACAATAAAA CAA TA GTCTTT ACGOTCATC CTC A GGA GG G G.ACCCAGAAATTGTACTTCACAGTTTTAATTGTGGAGGGGAATTTTTCT ACTGT A ATA CAA CA C AACT GT TTA ATAG TACT T G 3AAT A GTACTGA AGG G TC AAAT AA C A CT GO6AG GA AATGA C AC AAT CA CACT C CCATO6C AG A AT AA AA C A AAT T G T A A AC ATGTGGCAGGAAGTAGGAAAACAATGTATGCCCCTCCCATCAGT GGACAAATT A A AT GOT AT TAT CA AAT AT TA CAG GI3CT TACT AT T A AC A A GAG AT GB G'B B T G A A B AT A C AA CT A ATACTA C AGAG ATC CGBTC GA CA AG CT T C CCOG GAG0 CT TO GAAT TCT TT B AAGA CGA A A BGCCT CGTG ATACTCCT AT TT TT ATA GOT -56 TABLE GluAsnPheThrAspAsnAlaLysThrl lel leVaiHi sLeuAsnGlu SerValGinI leAsnCysThrArgProAsnTyrAsnLysArgLysArgl leHis IleGlyProGlyArgAlaPheTyrThrThrLysAsnl lel leGlyThrI leArg GInAlaHisCysAsnI leSerArgAlaLysTrpAsnAspThrLeuArgGlril le ValSerLysLeuLysGluGlnPheLysAsnLysThrlleValPheAsnGlnSer SerGlyGlyAspProGluI leValMetHisSerPheAsnCysGlyGlyGiuPhe PheTyrCysAsnThrSerProLeuPheAsnSerThrCysLysl leLysGlnI le IleAsrnMetTrpGlnGluValGlyLysAlaMetTyrAaProProI leGluGly GinI leArgCysSerSerAsnl leThrGlyLeuLeuLeuThrArgAspGlyGly LysAspThrAspThrAsnAsl: ihr TABLE

GATCTGAAAATTTCACAGACAATGCTAAAACCATAATAGTACACCTGAATGAA

,ACTTTTAAAGTGTCTGTTACGATTTTGGTATTATCATGTGGACTTACTT

TCTGTACAAATTAATTGTACAAGACCCAACTACAATAAAAG.AAAAAGGATACAT

AGACATGTTTAATTAACATGTTCTGGGTTGATGTTATTTTCTTTTTCCTATGTA

ATAGGACCAGGGAGAGCATTTTATACAACAAAAAATATAATAGGAACTATAAGA

TATCCTGGTCCCTCTCGTAAAATATGTTGTTTTTTATATTATCCTTGATATTCT

C" 'kGCACATTGTAACATTAGTAGAGCAAAATGGAATGACACTTTAAGACAGATA iTCGTGTAACATTGTAATCATCTCGTTTTACCTTACTGTGAAATTCTGTCTAT

GTTAGCAAATTAAAAGAACAATTTAAGAATAAAACAATAGTCTTTAATCAATCC

CAATCGTTTAATTTTCTTGTTAAATTCTTATTTTGTTATCAGAAATTAGTTAGG

TCAGGAGGGGACCCAGAAATTGTAATGCACAGTTTTAATTGTGGAGGGGAATTT

AGTCCTCCCCTGG,,GTCTTTAACATTACGTGTCAAAATTAACACCTCCCCTTAAA

TTCTACTGTAATACATCACCACTGTTTAATAGTACTGCAAAATAAAACAAATT

AAGATGACATTATGTAGTGGtGACAAATTATCATGAACGTTTTATTTTGTTTAA

ATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATTGAAGGA

TATTTGTACACCGTCCTTCATCCTTTTCGTTACATACGGGGAGGGTAACTTCCT

CAAATTAGATGTTCATCAAATATTACAGGGCTACTATTAACAAGAGATGGTGGT

GTTTAATCTACAAGTAGTTTATAATGTCCCGATGATAATTGTTCTCTACCACCA

AAGGACACGGACACGAACGAI2ACCGA

'TTCCTGTGCCTGTGCTTGCTGTGGCTCTAG

921130,p:\oper~ejb44O36-89.rsp,56 WO 90/03984 WO 9003984PCT/US89/04302 57 TABLE Me t L euiA r Pr oVa 161 uT h 'P r Th rA rg l ui 11 e L~s Lv~s Le u Asp G I vL e u TrAIa'ee~us~gl~ga as l 1esG1 ier L AsrIheThrAspAsriA aLvsThr Ile IleV alHi sLeiiAsriGluSe rV a IGl I leAsrCvsThrAir!Pro A snrg AsriLs A rLs A r51eHi s11eGlvF'rn Gl~9lPev~rTrv~nIeIlGIvh 1eAr~ AIaH Cv nIl~rAr.1av~F ns~-LuArGIr 1ez erv LeuLvsGlijG riF'heLsAsrLsThr IlIeV a Phe AsriG rI Se rEOe rlG l4 Asp FoGlu 11 eV a ltetHi sSerPhe AsiCvsG 2. G I v6 1 uF'hePheT v rCv!; As riThrSer~roLeuPhe AsriSeriThr'CvsLs I I eL s3 1 n-I eII e AsriMet TrPGInG1uVa I GwLsAa~etTwrA IaPro~ro I IeG IiG1jGln I Ie A r~ C~isSerSerAsrt I 1eThrG I LeuLeuLeuThrArAsp GlvG 1 Lvs A si:Th r A spThrAsriA spThrGlu I eArgArgG lriA aSer A r9G I u'LuG1'PheLeJ LvsThrLvsGl vProAr9AspThrPro I lePhe 1e61v WO 90/03984 WO 9003984PCT/US89/04302 58 TAB~LE

ATGTTACGTCCTGTAGAAACCCCAACCCGTGAAATCAAAAAACTGGACGGCCTG

TGGGCATTCAGTCTGGATCGCGAACGCGTGGCCGATATCATCGATGGATCTGAA

AATTTCACAGACAATGCTAAAACCATAATAGTACAtCTGAATGAATCTGTACAA

ATTAATTGTACAAGACCCAACTACAATAAAAGAAAAAGGATACATATAGGACCA

GO A AG A G C AT TTT TAT A C A AC AA A A A AT TAT A AT A G G A A CT ATA AA GA CA A AG CA CAT T GT AACATTAGTAGA GCAAAATGGAATG ACACTTTAAGA CAGA TAG TTAGCAAA

TTAAAAGAACAATTTAAGAATAAAACAATAGTCTTTAATCAATCCTCAGGAGGG

GACCCAGAAATTGTAATGCACAGTTTTAATTGTGGAGGGGAATTTTTCTACTGT

A AT ACAT CACCACTGTTT AAT ASTACTTGCAA AAT A AAACAAATT AT AAA CATB6

TGGCAGGAABTAGGAAAAGCAATGTATGCCCCTCCCATTGAAGGACAAATTAGA

TB OTT CAT C A A AT AT TA CA AGO BOBCT ACT TAT TA A C A A G A GAT G G TG G TA AG GO ACA C B.

BACACGAACGACACCGAGATCCGTCGACAAGCTTCCCGGGAGCTGGAATTCTTG

AAG A CGA AAGGBC CT CST G AT AC.T CCT AT TTTT AT AG ST 59 TABLE 6 LeuLysGluAlaValGlu Il3eAsnCysThrArgProAsriAsnAsnThrThrArg Ser leWisIleGlyProGlyArgAlaPheTyrAlaThrGlyAsaI leAlaGly AspI leArgGlriAlaHisCysAsnl leSerArgAlaLysTrpAsnAsnThrLeu LysGlralleVallleLysLeuArgAspGlnPheGluAsnLysThrl lel lePhe AsnArgSerSerGlyGlyAspProGlul leValMetHisSerPheAsnCysGly GlyGluPhePheTyrCysAsnSerThrGlnLeuPheSerSerThrTrpAsnGly ThrGluGlySerAsriAsnThrGlyGlyAsnAspThrl leThrLeuProCysArg I leLysGlul lel leAsnMetTrpGlnGluValGlyLysAlaMetTyrAlaPro Prol leLysGlyGlnValLysCysSerSerAsnl leThrGlyLeuLeuLeuThr ArgAspGlyGlyAsnSerLysAsnGlySerLysAsriThr TABLE 6A

CTGAAAGAAGCTGTAGAAATTAATTGTACAAGGCCCAACAACAATACAACAAGA

GACTTTCTTCGACATCTTTAATTAACATGTTCCGGGTTGTTGTTATGTTGTTCT

AGaTATACATATAGGACCAGGGAGAGCATTTTATGCAACAGGAGACATAATAGGA

TCATATGTATATCCTGGTCCCTCTCGTAAAATACGTTGTCCTCTGTATTATCCT

GATATAAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATAACACTTTA

CTATATTCTGTTCGTGTAACATTGTAATCATCTCGTTTTACCTTATTGTGAAAT

AAACAGATAGTTATAAAATTAAGAGACCAATTTGAGAATAAAACAATAATCTTT

TTTGTCTATCAATATTTTAATTCTCTGGTTAAACTCTTATTTTGTTATTAGAAA

AATCGATCCTCAGGAGGAGACCCAGAAATTGTAATGCACAGTTTTAATTGTGGA

TTAGCTAGGAGTCCTCCTCTGGGTCTTTAACATTACGTGTCAAAATTAACACCT

CtGPGGGAATTTTTCTACTGTAATTCAACACAACTGTTTAGTAGTACTTGGAATGGT

CCCCTTAAAAAGATGACATTAAGTTLGTGTTGACAAATCATCATGAACCTTACCA

ACTGAAGGGTCAAATAACACTGGAGGAAATGACACAATCACCCTCCCATGCAGA

TGACTTCCCAGTTTATTGTGACCTCCTTTACTGTGTTAGTGGGAGGGTACGTCT

C ATAAAAGAAATTATAAACATGTGGCAGGAAGTAGGAAAAGCAATGTATGCCCCT

TATTTTCTTTAATATTTGTACACCGTCCTTCATCCTTTTCGTTACATACGGGGA

CCCATCAAAGGACAAGTTAAATGTTCATCAAATATTACAGGGCTGCTATTAACA

GGGTAGTTTCCTGTTCAATTTACAAGTAGTTTATAATGTCCCGACGATAATiGT

AGAGATGTGGTAATAGCAAGAATGGTAGCAAGAATACAGA

TCTCTACCACCATTATCGTTCTTACCATCGTTCTTATGTCTCTAG

921130,p:\oper~ejb44036-89.rsp,59 WO 90/03984 WO 9003984PCr/ US 89/04302 TABLE 6B Mete 8rVal6 ~rr~ rGIuIIev~sejA j~l Tr h~re~rA!G Ar ~pe~s I es~sh~9r~nA tsih~rAr~rIIel ~j'c Ie ~pIIerGIrAIai Cy sr ~rr~av~pA .sih~uv~nIIe !1ev LeuAr A spGrF'heGuAsriLT hr e I ePhe s rESrSerG IVGlv As rGIuIIe etise.'e-ny~ GIv uPheFheTV, rCvs A riSer Thr G I rnL e 'Ph eSe rSe r ThrrT rp~Asr P' -ThrG 31 zGl1 ,,Se r As rt As rl Th~V rAph ~re~o ?,,.9IIevGIuIeIIeA Me~F1l~uaGIww etv ~or L:s ~n LwsCwsSe rSerAsril 1 eThrG2. vLeuLeuLeiuTh rA r-AsrG 1 vG I uA srSe T' Ltj snl~rw~nhOIu 9 e O ~u l Ph eLeuLvsThrLvsG1uFro 9sVFhrF~roI e~he I leG I '4 W4O 90/03984Pf/U8/40 PCr/US89/04302 61 TABLE 6C ATGTTAC GT CCTGTAG AA ACCCCAACCCGTG A AATCA AA A AACT GG AC GGCCT G T GO GCAT T CA G TCT GG AT C GC GAAC GC GT G GCC G AT CTG A A A G A A G C T GT A GA(- A ATTA ITT GT ACAAGGCCC AACAACAATACAACA AGAAGT AT A CAT AT A GG ACC A GGGAGAGCATTTTAT GCA ACAGGAGACATAATAGG AGAT AT A AG ACA AGCA CAT TG6T AAC AT TA GTAGAG CA A A A T GGA ATA AC ACTTT TA A A A CA A A TT TAT A A A A T T AA GAG A CCA AT T T GAG A A TAAA AC A A TA AT CTTT TA AT C G AT CC CT CA G GAG G A GACCCAGAA ATTGTAATGCACAGTTTTAATTGTG GAG GGG AAT TTTT CT ACTGT A ATT CA A C A C AACT G TT TA G TAG TACT TG GAAT GG TACT GA AG G G T C A A AT TA A C ACTG6GAG GA AATGA CA C AAT CA C CCTCCC ATGCA GA ATA A AAGiA AAT-T AT AAA C

ATGTGCAGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAAAGGACAAGTT

AAATGTTCATCAAATATTACAGGGCTGCTATTAACAAGAGATGGTGGTAATAGC

AAGAATGTACAAGAATACAGAGATCCGTCGACAAGCTTCCCGGGAGCTCGGAA

TTCTTGAAGACGAAAGG GCCTC*GTGATACTCCTATTTTT AT AGGT 62 TABLE 7 LeuAsnGluSerValGluI ieAsnCysThrArgProTyrAsnAsnValArgArg SerLeuSerlleGiyProGlyArgAlaPheArgThrArgGiul lel leGlyl le I ieArgGinAlaHisCysAsnlieSerArgAiaLysTrpAsnAsnThrLeuLys GinI leValGluLysLeuArgGluGlnPheLysAsnLysThrl leValPheAsn HisSerSerGiyGlyAspProGlul leValThrHisSerPheAsnCysGlyGly GluPhePheTyrCysAsnSerThrGlnLeuPheAsnSerThrTrpAsnGlyThr AspI leLysGlyAspAsnLysAsnSerThrLeulleThrLeuProCysArg le LysGinI lelleAsnMetTrpGinGiyVaiGiyLysAlaMetTyrAiaProPro I leGinGlyGinI ieArgCysSerSerAsnl leThrGiyLeuLeuLeuThrArg AspGlyGlyAsnSerSerSerArgGiu TABLE 7A

CTGAATGAATCTGTAGAAATTAATTGTACAAGACCCTACAACAATGTAAGAAGA

GACTTACTTAGACATCTTTAATTAACATGTTCTGGGATGTTGTTACATTCTTCT

AGTCTATCTATAGGACCAGGGAGAGCATTTCGTACAAGAGAAATAATAGGAATT

TCAGATAGATATCCTGGTCCCTCTCGTAAAGCATGTTCTCTTTATTATCCTTAA

ATAAGACAAGCACATTGTAACATTAGTAGAGCAAAATGGAATAACACTTTAAAA

TATTCTGTTCGTGTAACATTGTAATCATCTCGTTTTACCTTATTGTGAAATTTT

CAGATAGTTGAGAAATTAAGAGAACAATTTAAGAATAAAACAATAGTCTTTAAT

GTCTATCAACTCTTTAATTCTCTTGTTAAATTCTTATTTTGTTATCAGAAATTA

CATTCCTCAGGAGGGGACCCAGAAATTGTAACGCACAGTTTTAATTGTGGAGGG

GTAAGGAGTCCTCCCCTGGGTCTTTAACATTGCGTGTCAAAATTAACACCTCCC

GAATTTTTCTACTGTAATTCAACACAACTGTTTAATAGTACTTGGAATGGTACT

CTTAAAAAGATGACATTAAGTTGTGTTGACAAATTATCATGAACCTTAICATGA

GACATTAAAGGAGATAATAAAAATAGCACACTCATCACACTCCCATGCAGAATA

CTGTAATTTCCTCTATTATTTTTATCGTGTGAGTAGTGTGAGGGTACGTCTTAT

AAACAAATTATAAACATGTGGCAGGGAGTAGGCAAAGCAATGTATGCCCCTCCC

TTTGTTTAATATTTGTACACCGTCCCTCATCCGTTTCGTTACATACGGGGAGGG

ATCCAAGGACAAATTAGATGTTCATCAAATATTACAGGGCTGCTATTAACAAGA

TAGGTTCCTGTTTAATCTACAAGTAGTTTATAATGTCCCGACGATAATTGTTCT

'C3ATGGTGGTAATAGCAGCAGCAGGGAAGA

CTACCACCATTATCGTCGTCGTCCCTTCTCTAG

921130,p:\oper~eA44O36-89.rsp,62 WO 90/03984 PCY/US89/04302 63 TABLE 7B4 Me tLeu A rsFroV al1G I uThrF'roThr Ar9G I ul leL~vsL-ysLeu AspGl I Leu T rpA 1 a PheS e rL eu As A rgG Ii uA rgVa 1 A 1 a As P L eu As sriG1 uS er V a! 1 l1u Ile As ri C~sTh r A r s F'roT vrAssnP Ir As ri A Ar !Se rL euSe IrI e 01 2F' ro Gl~yAr A laF'heArThr A rGlu Ile I IeGI Ie I IeArgGrI nA IaHisC As nr I 1 e S er A~r A 1 a L~s T r A sr A sri Th r Le iiLvysO 1ri 1 1 e V I. C I uLvs L eu' A r9G I u~r nheLsAsrLsThr IlIeValPhe AsriHisSerSe rG1 jGl uAsp' F'r oG Iull eV Va 1 Th rHi sSe rF'he As sri Cv~sG I 0G1 j Glu Fhe F'he Tv r Cysa~As SerThrGlriLeuP heAs riSerThrTrA siGIThr As I lLsGYAsp AE r Lv~ne~re ~re~o-sA gIIe-sliIeIIesie TrPG IrsG1vV a16 1 Lds A laMetTvrAlaProF'ro I IeGlriGlvGln Ile A rg C,:sSe rSer As rtI I eTh rGlvLeuLeuLeuTh rA rg AspG G1 i AsriSe r'8e r SerArgG IuGIu I leA rArglriA IaSerArG uLeuG u'heLeLsTh r LqysOl vsF' roAr g A s pT hrPF r 011 e F'hell e 1 v WO 90/03984 pCT/US89/0 43 02 64 TABLE 7C AT GTT A CGT CC TOT A AAACCCCAACCCGT GAA AT CA AAAAACT G A CG GC CTG

TGGGCATTCAGTCTGGATCGCGAACGCGTGGCCGATCTGAATGAATCTGTAGAA

ATT AATT GT ACA A GACCCT ACAA CAATGT7A AGA AG0AA 0TCT AT CTAT AG G ACCA GGB AGA AG C AT TT CBTA CA AAGA GA A ATA A TAGGA AT TAT AAG BAC A AG C A C T7 TT A A CATT A 6T A A GCAAA ATGGAAT AAC ACTTT AAA A CAG6ATA OTT GA GA AATT A A G AGAA CA A TTT AAOAAT AAAAC AATAGOTC TTT AATCA TT CC TCA 0G0A GGGG A C C CA GAAATTG6T AACGCACABTTTTAATTGTGGABGGGA AT TTTT CTACT GI AAT TCAACACAA CT GTTTAATAOTACTTGAATGGT ACTG ACATTAA AGAGAT AAT AAA AATASC ACACTCATCACACTCCCATG CAG AATAAAA CAAATT AT AAAC ATG0

TGGCAGGGAGTAGGCAAAGCAATGTATGQCCCTCCCATCCAAGGACAAATTAGA

T B T TCAT CA A AT TATT TACAB BBCTBC TATTA AAC A AGAG AT 0 B TB BT A AT TABC AG C ABC AB GA AG AGAT CCBT CBA C A ABCITCCCGBA BC T GAAT TCT TGAA BA C AAA GBCCT CGTG AT ACT CCT ATTTTT ATAG GT WO 90/03984 r'iU8/40 PCUUS89/04302 Table 8 CTGA-ACCAATCTGTAGAAA'TTAATTGTACAAkGACCCAAC GACTTGGTTAGACATCTTTAATTAACATGTTCTGG GTTG

AACAATACAAGAAAAAGTATCCGTATCCAGAGAGGACCAGGGAGAGCATTTGTT

TTGTTATGTTCTTTTTCATAGGCATAGGTCTCTCCTGGTCCCTCTCGTAAkACAA ACATAGGAAAATAGGPATATGAGACAA.GCACATT GTAACATTAGTAGAGCA

TGTTATCCTTTTTATCCTTTATACTCTGTTCGTGTAACATTGTAATCATCTCGT

AAATGGAA~TAA CACTTTAAACAGATAGATAGCAAATTAAGAGAACA-ATTT GGA TTTACCTTATTGTGAAATTTTGTCTATCTATCGTTTAATTCTCTTGTTAAA CCT AATAATAAAACAATAATCTTTAAGCAGTCCTCAGGAGGGGACCCAGAAAtTTGTAml- TTATTATTTTGTTAT7TAGAATTCGTCAGGAGTCCTCCCCTGGGTCTTTAACAzT ACGCACAGTTTTATTGTGGAGGGGAATTTTTCTACTGTP TTCAACACACT G

TGCGTGTCAAAATTAACACCTCCCCTTAAAAAGATGACATTAAGTTGTGTTGAC

TTTATAGTACTTGGTTTATAGTACTGGAGTACTAAAG GGTCAAATAA CACT AAATTATCATGAACCAA.ATTATCATGAACCTCATGATTTCCCAGTTTATTGT GA GAGGAAGTGA CACAATCA CCCTCCCAT CCGAATAAAACAAATTATAAA CAT G

CTTCCTTCACTGTGTTAGTGGGAGGGTACGTCTTATTTTGTTTAATATTTGTAC

TGGCAGGAAGTAGGAAAAGCAATGTATGCCCCTCCCATCAGTGG%-ACAAATTA

GA

ACCGTCCTTCATCCTTTTCGTTACATACGGGGAGGGTAGTCACCTGTTTAATCTI~r TGTTCATCAAAIWASTACAGGGCTGCTATTAACAAGAGATGGTGGTAATA

GCAAC

ACAAGTAG-TTTATAA.TGTCCCGACGATAAkTTGTTCT CTACCACCATTA.T- OTGT G AATGAkGTCCGA 38

TTACTCAGGCTCTAG

66 TABLE 9 EEl-707-1

MN

EEl-708-1 KW4 -4-1 JH3 WMG3

BAL

EE3-7-3 KW4-6-1 EE3-4-3 EE7-3-1 EE7 -15-3 EE7-24-1 EE6-4-1 JG1 DD3-1 DD7-1 EEl-330-1 Sc DD10-1 WMJ1. 5 KW3-2-2 DD4-1 AFL3O-4-1 KW2-1-1 KW2 -9-1 EE6-4-4 EE7-20-3 RJS426 KsW4 -13-1 AFL30-6-2 KW2-8-2 -16-1 KW2-8-1 EE5-6-1 EE6-3-5 TM5-8-1 TM5-12-1 EE5-3-2 EE3-2-4 1 WH331 EEl-279-1 EE3-5-1 WH721 KW4-3-2 TM4-1i-1 TM5-13-1 SF2 SF4 Z321 WMJ2 EE6- 1-1 KW4-1O0-1 TM5-7-1 CTRPNNY TRK RIHI CTRPNYN KRK RIHI CTRPNNN TRR RIHI CTRPNNN TRR RIHI CTRPSKT TRR RIHI CTRPNDI ARR RIHI CTRPNNN TRK SIHI CTIPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN TRK SIHI CTRPNNN GIHI CTRPNNN TRX GIHI CIRPNNN TRK GIHI CTRPNNN TTR SIHI CTRPNNNVRRR HIHI CTRPNNNVRRR HIHI CIRPHNTI RR RIHI CTRPYSNV RN RIHI CTRPYSNV RN RIHI CTRPY6NV RN RIHI CTRPYSNV RN RIHI CTRPYSNV RN RIHI XTRPYSNV RN RIHI CTRPYNKK RIRHMHI CTRPNNKIPR HFHI XXRPNNY TRK GIHI XTRPNNN TRK GIHI CTRPNNN TRK. GIHI CTRPDNN ARK GIHI CTRPDNN ARK GIHI XTRPNNN TKK GIHI CTRPNNN TKK GIHI CIRPNIN TGK RIPI XTRPNNN TRK SIPI XXXPSNN TRK SIPI XXXXXXX XRK SIPI CTRPNNN TRK RISI CMRPNNN TRK SINI XXXXNNN TRK SINI CTRPNNN TRK SIAI CTRPNNN TRK RIYI CTRPNNN TRK RIYI CTRPNNN TRK SIYI CTRPNNN TRK SIY At CMRPNNN TRK SISI CTRPYNNV RR SLSI CTRPNNN TRK RIKI CTRPNNN TKK GIYI CTRPNNN TKK GIYI GFGRAFY TTGQVIGRI GPGRAFY TTKNIIGTI GPGRAFY TTGQVIGRI GPGRAFY TMGQIIGDI GPGRAFY ATGDIAGDL GPGRAFY T GKIIGNI GFGRAFY TTGEIIGDI GPGRAFY TTGEIIGDI GPGRAFY TTGEIIGDI GPGRAFY TTGEIIGDI GFGRAFY TTGEIIGNI GPGRAFY TTGEIIODI GPGRAFY TTGEIIGDI GPGRAFY ATGAIIGDI GPGRAFY ATGDIIGDI GPGRAFY ATGDIIGDI GPGRAFY ATGDIIGDI GPGR.AFY ATGDIIGD! GPGRAFY ATGDIIGDI GPGRAFY TGEIRGNI GPGRAFYY GEIRGNI GPGRAFS TTRGIQGDI GPGRAFH TTKRITGDM GPGRAFH TTKRITGDM GPGRAFH TTKRITGDM GPGRAFH TTKRITGDM GPGRAFH TTKRITGDM GPGRAFH TTKRITGDM GPGRAFY ATG GMGDI GPGRAFY ATGGIEGDI GPGRAIY ATGDIIGDI GPGRAVY TTGRIVGDI GPGRAVY TTGRIVGDI GPGRAVY TTGRIIGDI GPGRAVY TTGRIIGDI GPGRAWY TRTRIIGDI GPGRAVY TARRIIGDI GPGRAFY TTGAIKGNI GPGRAFY TTGEIIGDI GPGRAFY ATGDIIGNI GPGRAFY ATGEIIGDI GPGRAFY ATRQIVGDI GPGRAFY TTGQIIGDI GPGRAFY ARGEIIGDI GPGRAFY ATRRIIGDI GPGRAFY ARQQIIGDI GPGRAFY ARQQVIGDI GPGRAFH TTGRIIGDI GPGRAFH TTGRIIGDI GPGRAFF ATGDIIGDI GPGRAFR YRE IIGI GPGRAFV TTKQIIGDI GPGRAVY TTEKIIGDI GPGRAVY TTEKIIGDI

RQAQC

RQAHC

RQAQC

RKAHC

RQAHC

ROAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAYC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAYC

RQARC

RQARC

RQARC

RQARC

RQARC

RQARC

RQAHC

RKARC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RRAXX

RQAHC

RQAHC

ROAHC

RKAHC

RKAHC

RQAHC

RQAHC

RQAYC

RRAHC

RRAHC

5' I I

'SI,

s~ 921130,p:\opereiA4O3689xsp,66 67 TABLE 9 (Continued) EE7-~15-1 DD-3-3 5-3 KW3 -9-2 JG2 EE3-5-2 AFL30-1-3 AFL30-3-1 KW2-2-2 EE5-3-3 EE5-6-3 EE5-1O-3 EE5-11-3 EE7-24-3 KW4 -3-1 KW4-2-2 WH425 WH7 18 WH244 EE3-6-2 EE6-3-1 DD9-1 TM5-11-1 EE5-1O-5 -11-1 EE5-14-1 KW2 -6-1 DD 5- 1 TM4-7-3 AFL30-11-1 AFL30-12-1 KW4-12-1 KW4 -12-2 KW4-7-1 KW4-7-2 Kw3-6- 1 IIIB (BH1O)

LAV-BRU

EE5-1O-1 E7-3-3 EE7-6-2 KW2-1-3 EE3-6-1 EE7 -15-2 EE5-3-1 TM4-14-2 KW3-6-2 RF' 6TM3-1-1:

RF

EE7-20-1 WLC451 TM3-9-4 EEl-- 706--i CTRPNNN TKK CTRPNNN TKK CTRPNNN TRK CIRPNNN TRK CTRPNNN~ TRK C.MRPNNN TRK XTRPNNN TSR C;TRPNNN TSR CTRPNNN TSR XTRPNNN TSR CTRPNNN TSR CTRPNNN TSR CTRPNNN TSR CTRPNNN TSR CTRPNNN TKR ,CTRPNNN TKR XXXXNNN TRR XXXXXXX XSK XXXXNNKI 1(1R CTRPNNN TRK CTRPNNN TKK CTRPNNN TRR CTrRPNNW, TQK Cm.RPNNN TRK CTRPNNN TRI( CTRPNNN TRK CTRPNNN TRK XXXXNNN TRA CIR"t1NN TRK CTRPNNN TKK CTRPNEN TKK CTRPNEN TKFK CTRPSNN TK(.

CTRPSNN TKR CTRPNNN TRK CTRPNNN TRK CIRPNNI TRR CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRK CTRPNNN TRR CTRPNNN TRK CTRPNNN TRK CTRPNNH TEK CTRPNNH TPIK CTRPNNN TRK CTRPNNN TRK

GIRI

GIRI

GIRI

RIGI

SINI

SINI

GIRI

GIRl CI RI

GIRI

GIRI

GIRT

GIRl

GIRI

GIRI

GIRI

sisi

SISI

RIHI

GIHI

GIHI

SIHI

RITI

GIFI

GIFI

GIFI

GI FI R Lls1

AMSI

C1AI SLY I

SLYI

SLYI

9L~yi

GIHM

GIHM

SMSM

GPGRAVY

GPGRAVY

GPGRAIY

GFGRAIL

GPGRAFY

FPGRALY

GPGRAI L GPGRAI L

GPGRAIL

GPGRAI L GPGRAI L GPGRAI L GPGRAI L

GPGRAIL

GPGRAVY

GPGRAVM

GPGRALY

GPGRAWY

GPFRPFY

GPGRTFY

GPGRKWI

TAQRI IGDI TARGI IGDI

ATARIIGDI

00 ENIC-DI

ATGQIIGNI

TTGQIIGDI

ATERIIGDI

ATERI IGDI

ATERIIGDI

ATERIIGDI

ATERI IGDI ATERI IGDI

ATERIIGDI

ATERIIGDI

ATDRI IGDE QQTRI IGDI

TTGAIIGSI

QQEEVIGDI

TTK IGDI ATGAI IGDI

TRTKIIGDI

GPGRWSVHTTGEIVGD I

GPGRVFY

GPGRNIY

GPGRV4Y G3? (AVI Yr

G)PGRSFY

JJPGRKLY

GPGRTLY

GPGRRFH

GPGRRFH

GPGRRFH

GPGRRFH

GPGRAFY

GPGAAFY

GPGRAFV

.7 p."

SIRIORGPGRAFV

SIHIQRGPGRAFV

SIRIQRGPGRAFV

S IRIORGPG.AT-V S IRIQRGPGRAFV

RIRIQRGPG-?AFV

RIRIQRGPGRAFV

KIRIQRGPGRAFV

KIRIQRGPGRAFV

KIVSRGGPGRAFV

GIRV GPGRAVY RITK GPGRVIY SITK GPGRVIY RITL GPGRVLY RVTL GPGRVWJY RVTM GPGRVWY RITM GPGRVYY

TTGKIVGDI

TTGNI IGDI TTGNI IGDl TTGNI IGDI TTGNI IGDI

ATRNIVGDI

TRN4KI IGDI

AREKIIGDI

VTKAITGDI

VTKAITGDI

AITGDI

wiPKAITGDI TOENI GDI TQENI GDI ATRQI IGDI TIGK IGNM TIGK IGNM TAGK IXNM TIGK IGN4 TIGK IGNM TIGK IGNM TIGK IGNM TIGK IRNM TIGK IRNM TIGK IRNM

STDKIIGDI

ATGQIIGDI

ATGQIIGDI

TTGRI IGDI

TTGEILGNI

TTJGEIVGDI

TTGQTNI

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

ROAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHiC

RQAHC

RQA!HC

RQAHC

RQAHC

RQAXX

xxxxx

RQAYC

RQAHC

RQAHC

RQAHC

RQAHC

RKAHC

RKAHC

RKAHC

RKAHC

RQAHC

RQAHC

RQAHC

RQAHiC

RQAHC

RQAHC

RQAPC

RQARC

RQAHC

RRAHC

ROAHC

RQAHC

RQAHC

RQAHC

RQAHC

1EEQAHC

RQAHC

RQAHC

RQAMC

RQAhC

RQAHC

RKAHC

RKAHC

RRAHC

ROAHC

RQUIC

'AQAHc 921130,p:VWpe\ch44036-89.rsp,G7

S.

4.- 68 TABLE 9 (Continued) EEl-706-1

BBVA

KW2-1-2 EEl-317-1 SF170 KW2-6-2 Z3 EE3-8-1 RW4-8-1 KW4-8-3 TM4-14-1 KW2-2-1

LAV-MAL

EEl-317-2 EEl-707-2 SF33 W~l-21-3 TM4- 14-3 EE7-6-1 DD1 -1 AFL30-5-1 EE7-21-1 TM4- 13-2 TM4 -13-3 TM3 1 AFL30-9-1 AFL30-9-2

LAVELI

jyl Z6 \e.TRPNNN TRK RITM CT1RFNNN TRK RITM CTRPNNN TRK RITM CTRPNNN TRK GIHI CTRPNNN TRK GGTI CTKPNNN TKK GIFI

CTRPGSDKKIRQSIRI

CIRPNNN TRK SIPM CTRPNNN TRK SIPM CTRFNNN TRK SIPM CTRPNNY ARR GIRV C'TRPNNN TEK ESYC C'FRPGNN TRR GIHF CTRPNNN IRK RITR CTRPNNN IRK IRITR CTRPNNN RRR RITS CTRPNNN TRR RIHI CTRPNNY AKR GIRV CTRPGNN TRR S151 CTRPNNN TflK SIRI CTRPN1NN TRK SIRI CTRPNNN TRK SIRY CIRPNNN TKR AMY CTRPNNN TKR AMY XIRPNNN TRK GIYV CIRPNNN TRK GIYV CIRPNNN TRK GIYV CARPYQN TRQ RTPI CTRPDNKITRQ STPI CTRPYKN TRQ STPI

GPGRVYY

GPGRVYY

GPGRVYY

GPGT FY

GPGQAFY

GPGKNIY

GPGKVFY

GPGKAFY

GF -AFY G -,KAFY

GPGSATY

?RGPGQAFV

GY'GQALY

GPGKV IY

GPGKVIY

GPGKVLY

GPRRAFY

EPGKAI I TAGQI IGMI TTGQI IGDI TTGQI IGNI TTGEI IGDI

ATGDIIG~DI

TTENI IGDI

AKGGITG

ATKEI IGDI TTGDI IGDI ATGDI IGDI TAPS IIADI TIGK IGNM

TTGIVIGDI

ATGQI IGDI ATGQI IGDI TTGEI IGDI

TTGQVIGRI

ATKKIIENI

RQAHC

RRAHC

RQAHC

RQAHC

RQAYC

RKAHC

QAHC

RQAHC

RQAHC

RQAHC

SQAHO

RQAHC

RRAYC

RKAHC

RKAHC

RKAYC

RQAQC

KKAHY

RQAHC

RQAHC

RQAH-C

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

RQAHC

GQAHC

RQAYC

GQAHC

RPERAFFTOTGDVIGDI

GAGRAIY

GAGRAIY

GAGRAIY

GQGRAIH

GQGRAIH

GSGRAVY

GSGRKVY

GSGRKVY

GLGQS LY

GLGQALY

GLGOALY

ATARI IGDI ATERI IGDI ATARI IGDI

TTDRIIGDI

TTDRI XGDI

TRDKIMGDI

TRHKI IGDI TRQKI IGDI TTRSRS II TTR IKGDI TTRGRTKI I *iIa U, I U

I

I

4- 44(4

C

44 44$ I C 4 4.

C.

-I 4.

4 V

'S

4 *44 444 4 4 4 4 4 I.

4.

921130,p:~cper\eAh44036489.rsp68 Table X13 X 12 xii Xlo 1(9 X8 1(7 X6 5 4 Y3 X2 I 120 Ii 1.10 Li JJU T 115 1 11 M 3 AlI R 129 1 1 K I P 132 N115 Y 10 S 4 D 3 G03 H I N 121 S 7 K 2 E 2 y I D I I1 I 01I N 118 NV 2 Y 4 K1 3 1 2 T 2 DK 1 H I K 1 T 113 K 3 V 7 1 3 AS5 R 3 ElI p 1 R 103 K 18 8 9 1 2 E 2 0 1 T I K 90 R 34 1 1 N 6 Q 4 Al1 IR I RQ 1 L 6 M 3 T 3 V 2 01I FlI y I El1 R 28 T 14 S 10 P 10 FS5 N 4 A 2 K I 01I v I R 2 M 11 V 6 IOR 9 L 2 K 2 F l S I G01 y1I SRG 1 YQR I ElI R I L 3 A 3 S 3 Q 2 ElI R I Y2 YL Y4 Y5 Y6 Y7 Y8 Y9 Yi0 Yui Y12 Y13 Y14 Yis Y16 Y17 A 1.11 rijio 0130 I 119 106 V ID R.171 0 5 S I T I A 104 V 14 N 5 R 4 K 4 T 2 F 1 p 1 WI1 F 75 1 25 V 15 L 8 w 5 YS5 0 1 Si1 T I Y 94 V 13 H ?4 L 8 Fl S52 1 1 TI1 R I Fr I T 102 R 11 1 11 0 5 A 5 mlI O 78 E 17 K 14 R 10 T 3 P.5 Q04 A 3 HI1 Ni1 p1I R 34 K 23 0 20 E 20 D 13 N 9 A 8 G04 S32 1 1 1 115 V 7 R 2 N I G01 1 105 T 12 V 7 K 2 M 2 R 2 L I 51I El1 Q01 A 1 R 3 ElI K I AlI N 23 1 3 R 3 Ti1 S I 1 169 ME 16 G02 K 1 S I El1 K 16 R 6 Y 8 RB8 Q03 70 TABLE 11 NPLE f NAME MEI-707-1 w4 KEi-706-1 N4 -4-1 J3 NNJ3

RAL

E-E3 -7-3 30"4-6-1 EE3 -4-3 EZ7-3 -1 XM7-15-3 ZE7 -24-1 zzE -4-1 J03.

003- L D07- 1 EI -330-1 sc 00D10-1 304315 1013-2-2 D04-1 "FL3 0-4-1 3042-1-1 1042-9-1 P-E 6-4-4 3V.E7-20-3 AJS426 1044-13-1 AFL30-6-2 3012-S-2 1145-16-1 3042-8-1 EzS -6-1 EE-3-5 1145-3-2 T"45-22-1 EKS -3-2 XXN3-2-4 W8331 Ezi-279-1 mz3-5-1 1lE721 K4 -3-2 r141-1- TMS-13-1 SF2 SF4 Z321 NW42 EKE--i 3044-10-1 1145-7-1 E7-iS-i 003-3 AFL30-5-3 3013-3-2 J62 E.E3 -5-2 AFrL3O -1-3 ArL3O-3-1 102-2--2 zE5-3-3 XZS-6-3 ZS-1-3 xzE-ii-3 EZ7-2 4-3 1014-3-i 1014-2-2 R4 25 MR7is U82 44 ZE3-6-2 TRUAHIGPGAAFYTrG r 4 4AIHI GPG RAFYVG K K I- S S A-- S

Q

G A-

A--

A--E

A-RH of A-A V-A H- V-N N--K V-N N--K V-N N--K V-N N--K V-N AIAHF A--

V--

-K-G -R

A-A

ARO

Y- -A K V--K -K-G-Y -1-0-A V--AG TK-G-A _AA

AA

G--

S-N- 0-A-

S-U-

-SAG-A ILA-Z

ILA,-&

-SAG-I ILA-Z

ILA-Z

-SAtG_----ILA-Z -SRG-R ILA-& -SAkG-A------ILA-l -ZAG-A _V-A-D -ZAG-A VM-NOO AS-s- L_ IS-u-S- -3t -K

A

TYPE (0TOTAL) (2/3)

P

(3/3) (3/3) (2/2) (3/3) (1 /1) (2/3) (1/3) (213) (2/3) (1/7) (116) (3/3) (5/5) (3/3) (6/6) (5/5) (1/3) (1/1) (1/3) (1/3) (3/3) (4/6) (2/4) (1/4) (1 (1/2) (2/3) (3/7) (3/3) (1/3) (3/3) (direct) (3/3) (1/2) (direct) (2/3) (5/8) (3/8) (3/3) (3/3) (3/3) (1/3) (3/7) (1/5) (2/2) (1/2) (2/2) (1/1) (2/2) (1/3) (1/2) (1/3) (1/2) (1/3) (1/3) (2/2) (direct) (direrit) Idirect) (1/3) (1/3) 0 0 0 RTHIGPGRAEY 0 0 0 0 0 0 0 0 0 0 0 0 HGGA 0 0 0 TIGG&F 0 0 0 e 1~TRTGPGA 1 HIGPGRAi 1GCM 2 1ao 2 2 2 2 HGPA 1 2 2 1 1 2 1 1 2 2 2 2 MISMATCNES (5 TO 13) ft. C ft...

ft ft UftIft 4 ft 14 ft ft ft ft ft ft.

ft. ft ,ft C ft ft ft ft 70A 196:3-1 TNhs-1~' -1 115-11-1 XE5-14-1 DDS-1 TN4-7-3 NY 5 AFL30-11-1 AFL30-12-1 flN4-12-1 30(4-12-2 1014-7-1 30(4-7-2 3013-6-1 iiia(BHIO)

LAV-BRU

Z15-10-1 117-3 -3 X17-6-2 3012-1-3 113-6-1 117-15-2 ZE5-3 -1 T?4-14-2 1013-6-2

RF

117 -2 0-1 CDC451 XZ1-706-1 XEI-706-2

BAVA

1312-1-2 11l-317-1 SF170 3012-6-2 113-S-1 KM 4-S-1 304-1-3 114-14-1 W0(-2-1 LA V-MAL M1-317-2 111-707-2 SF33 117-21-3 T34-14-3 XE7-6-1 001-1 AFL3O-5-1 XE7-21-1 1144-13-2 1144-13-3 TM3-7-1 AFL30-9-1 AFL30-9-2

IAVT.LI

ITT'

f-L-ARE -K-SLY- -N-V-K -URSLY- l-N-V-K -KOSLY- -N-V-K -mRLY- -N-V-K G--M 0E ASHEN-- VA-A KsI-Q-- V-I- KSI--V V-I- KSI--V V-1- KSI--2,R V-I- KSI-Q-- V-I- KI--ORG---v

VI--

VI--

G-AV TMC-

G

-T03-- -Y-IfiT

T-GE-

SO- K1---E- A-A0-AV S-T--AP ZKISYQR 0--V-I- I Tf KVI-A-- I TR

KVI-A--

fl-A--I'S AICAC-AV K-IIA-K flS-S- A-E F-CT S-fl- I-A-A S-fl- I-A-E S-AT -A--IAA -KAA-Y- IH--O -KRA-Y- IH--D 0G-TV V--AD 0G-TV -KXV--AN 0G-TV -KV--AQ OSL fl VETP- (0-L T OST?- Q-L AR 2 INIOPGR 2 3 3 3 3 4 3 3 3 3 1 1 GPGP.A~ 3 2 2 2 2 2 2 2 2 2 3 GPGRAF 3 GP-9' 4 4

THGPG~

IHIpG; 4 C 4* 44 4 4 4 4 4 4 4 44,4 (144 (4 4* 4 4 4 4 4 4- 41(4 4 4 4 4 4 4444 4 4 6 4* 4 4 444 4 4 3 3 4 3 PCf/US89/04302 WO 90/03984 71 Table 12 COMMON SEQUENCE PATTERNS SEOUENCE APPROXIMATE OCCURENCEM(' I a IG PGR 1a IG P GRA Ia I GP GR AF 32 IGPGRA 52 GPGRAF IGPGRAF IGPGRAaY 31 WO 90/03984 PCIIUS89/04302 72 Table 13

IIIB

MetThrArglleGlnArgGlyProGlyArgAlaPheVaI GlyGlyGlyGly-

RF

SerfleThrArgGlyProGlyArgVallleTyr GlyGlyGlyGly ArglleHislleGlyProGlyArgAlaPheTyr GlyGlyGlyGly SerfleHisIIeGlyProGlyArgAlailheTyr GlyGlyGlyGly- WMJ 1 Hisl eHisl eGlyProGlyArgAlaPheTyr GlyGlyGlyGly LcuSerllleCys WO 90/03984 WO 90/03984PCV/US8 9/04 302 73 Table 13A E.coli BG MetLeuArgProValGluiThrProThrArgGlulleLysLysLeuAspGlyLeuTrp- AlaPheSerLeuAspArgGluArgValValArg-lyrHisArgTrpIlet'rgGlnAlaSer-

IIIB

MetThrArglleGlnArgGlyProGlyArgAlaPheVal GlyGlyGlyGly

RF

SerlieThrArgGlyProGlyArgVallleTyr GlyGlyGlyGly-

MN

ArglleHislleGlyProGlyArgAlaPheTyr GlyGlyGlyGlys: SerI~eHisIeGlyProGlyArgAlaPheTyr GlyGlyGlyGly- WMJ1I HislleHislleGlyProGlyArgAlaPhel'yr GlyGlyGlyGly LeuSerlleCys

Claims (71)

1. A compound having the capability of eliciting, and/or binding with, HIV neutralizing antibodies, said compound having the formula: ax G z Gyb wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an FIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant and wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T.

2. The compound, according to claim 1, wherein' said compound is circularized.

3. The compound, according to claim 1, wherein said compound comprises epitopes from the principal neutralizing domain of more than one HIV variant.

4. A polypeptide selected from the group consisting of HIV 10 Kd fusion protein denoted Sub 1 as hereinbefore defined; t HIV protein portion of Sub 1 capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; HIV 18 Kd fusion protein denoted Sub 2 capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; HIV protein portion of Sub 2 capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; S. 921130,p:\ope\ejh44036-89sp,74 c= ro^ 75 HIV 27 Kd fusion protein aenoted PBlRF capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; HIV protein portion of PBlRF capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; HIV 28 Kd fusion protein denoted PBlMN as hereinbefore defined: HIV protein portion of PB1MN capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; HIV 26 Kd fusion protein denoted PBlSC as hereinbefore defined; HIV protein.portion of PBlsc capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; (11) HIV 26 Kd fusion protein denoted PB1WMJ2 as hereinbefore defined; (12) HIV protein portion of PB1WMJ 2 capable of eliciting and/or binding with neutralizing antibodies as hereinbefore defined; (13) peptide IIIB(BH10)-PND as hereinbefore defined; (14) peptide RF-PND as hereinbefore defined; peptide MN-PND as hereinbefore defined; (16) peptide SC-PND as hereinbefore defined; (17) peptide WMJ-2-PND is hereinbefore defined; (18) peptide LAV-MAL-PND as hereinbefore defined; (19) peptide SF-2-PND as hereinbefore defined; peptide NY5-PND as hereinbefore defined; (21) peptide Z3-PND as hereinbefore defined; (22) peptide WMJ1-PND as hereinbefore defined; (23) peptide WMJ3-PND as hereinbefore defined; (24) peptide Z6-PND as hereinbefore defined; peptide LAVELI-PND as hereinbefore defined; (26) peptide CDC451-PND as hereinbefore defined; (27) peptide CDC42-PND as hereinbefore defined; (28) peptide BAL-PND as hereinbefore defined; 921130,p:\oper\ejh44036-89.rsp75 76 (29) peptide HIV-2-PND as hereinbefore defined; peptide 135 as hereinbefore defined; (31) peptide 136 as hereinbefore defined; (32) peptide 139 as hereinbefore defined; (33) peptide 141 as hereinbefore defined; (34) peptide 142 as hereinbefore defined; peptide 143 as hereinbefore defined; (36) peptide 131 as hereinbefore defined; (37) peptide 132 as hereinbefore defined; (38) peptide 134 as hereinbefore defined; (39) peptide 339 as hereinbefore defined; RP342 (WMJ2) as hereinbefore defined; (41) RP343 (SC) as hereinbefore defined; (42) RP60 (IIIB) as hereinbefore defined; (43) RP335 (IIIB) as hereinbefore defined; (44) RP337 (IIIB) as hereinbefore defined; RP77 (IIIB) as hereinbefore defined; (46) RP83 (WMJ1) as hereinbefore defined; (47) RP79 (IIIB) as hereinbefore defined; (48) RP57 as hereinbefore, defined; (49) RP55 as hereinbefore defined; RP75 as hereinbefore defined; 4,(51) R-P56 as hereinbefore defined; (52) RP59 as hereinbefore defined; 5 3 R P 7 4I I F s h r i n e e d f n d RP74 (IIIBRFN) as hereinbefore defined; (54) RP80 (IIIBRFN) as hereinbefore defined; RP81 (IIIB,RFMN) as hereinbefore defined; (56) RP82 (21FWMJ1,MN) as hereinbefore defined; (57) RP137 (WMJ1,MN) as hereinbefore defined 4(59) RP140 (IIIB,RF)..as hereinbefore defined: peptide 64 (HIV-IIIB/HIV-RF/HIV-MN/HIV-SC) as (61) peptide 338 (HIV-IIIB/HIV-RF) as hereinbefore defined; (62) peptide 138 as hereinbefore defined; (63) RP342 as hereinbefore defined; (64) RP96 as hereinbefore defined; 921130,pAopex~eAh44O36-89.rsp76 77 RP97 as hereinbefore defined; (66) RP98 as hereinbefore defined; (67) RP99 as hereinbefore defined; (68) RP100 as hereinbefore defined; (69) RP102 as hereinbefore defined; RP88 as hereinbefore defined; (71) RP91 as hereinbefore defined; (72) RP104 as hereinbefore defined; (73) RP106 as hereinbefore defined; (74) RP108 as hereinbefore defined; RP70 as hereinbefore defined; (76) RP84 as hereinbefore defined; (77) RP144 as hereinbefore defined; (78) RP145 as hereinbefore defined; (79) RP146 as hereinbefore defined; RP147 as hereinbefore defined; (81) RP150 as hereinbefore defined; (82) RP151 as hereinbefore defined; (83) RP63 as hereinbefore defined; (84) RP41 as hereinbefore defined; RP61 as hereinbefore defined; (86) RP75 as hereinbefore defined; (87) RP111 as hereinbefore defined; (88) RP113 as hereinbefore defined; (89) RP114 as hereinbefore defined; (90) RP116 as hereinbefore defined; (91) RP120 as hereinbefore defined; (92) RP121c as hereinbefore de.ined; (93) RP122c as hereinbefore defined; S(94) RP123c as hereinbefore defined; and chemical modifications thereof capable of eliciting S S and/or binding with neutralizing antibodies. A DNA sequence which codes for a polypeptide, other than the naturally occurring HIV envelope protein, said polypeptide having the capability of eliciting, and/or binding with, neutralizing antibodies, said polypeptide 921130,1\oper\ejb,44036-89.r,77 ^.0rr. 78 comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant wherein said polypeptide has the formula ax G z G y b wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant and wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T.

6. A composition capable of eliciting and/or binding with neutralizing antibodies to a broad range of HIV variants, said composition comprising a pharmaceutically acceptable carrier and/or diluent and one or more compounds having the following formula: ax G z G y b wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; and z is P, A, S, 0, or L; and a, b, x and/or y is from a peptide of the HIV principal neutralizing domain as hereinbefore defined; and either a or b, but not both may be omitted; either a or b "I individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant, and wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T. 921130,p:oprejh,44036-89.rsp,7 79

7. A composition according to claim 6, wherein x is selected from: 2c(O) is not present X(l) aX x(2) x 2 x 1 x(3) x 3 x 2 xl x(4) x 4 x 3 x 2 xl x 5 x 4 x 3 x 2 xl x(6) x 6 x 5 x 4 x 3 x 2 xl sx 7 x 6 x 5 x 4 x 3 x 2 F-l x(8) x 8 x 7 x 6 x 5 x 4 x 3 x~xl x(9) ,cgx 8 x 7 x 6 x 5 xc 4 x 3 x 2 x 1 xl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl X( 11) =xllxlOxgx 8 x 7 x 6 x 5 x4x 3 x2xl x( 12) xl 2 xllxlQx 9 x 8 K 7 x 6 x 5 x 4 x 3 x 2 xl x( 13) xl 3 xl 2 xllxl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl y(O) Sy is not present yMl Yi y(2) y2yl y(3) y3y2yl y( 4 ay4y3y2yI. y5y4y3y2yI y(6) y6y5y4y3y2y1 y(7) y7y6y5y4y3y2y2, Y(9) ygy8y7y6y5y4y3y2y1 Yl0y9y8y7Y6y5y4y3y2y1 Y(11) YIiYOy9yBy7y6y5y4y3y2y1 y(1,2) Yi 2 YliylOy9y8y7y6y5y4y3y2y1 Y(13) Y13y12y11yIOy~y8y7y6y5y4y3y2y1 y(14) Y14Y13yl2y11y1Oy9yBy7y6y5y4y3y2y1 Yi 5 yi 4 yi3yl2yllylOy9yBy7y6y5y4y3y2y1 y(16) Yl 6 yl 5 yl4yl3yl2yllylOy9y8y7y6y5y4y3y2yI y( 17) Yi 7 yi6yi5y14y13y12Y1 iyiOy9yBy7y6y5y4y3Y2Y1 and wherein 4* a i 44 4 4 xi is X 2 is X 3 is 1, R, M, IQR, V, L, K, F, S, G, Y, SRG, or YQR; H, R, Y, T, S, P, F, N, A, K, G, or V; I, L, M, T, V, E, G, F, or Y; 921130,p:\oper\eAb44036-89nsp,79 80 N 4 is R, S, G, H, A, K, or not present; Xis K, R, I, N, Q, A, IR, RQ, or not present; x 6 is R, K, S, I, P, Q, E, G, or T; X 7 is T, K, V, I, A, R, P, or E; x 8 is N, NV1, Y, KI, I, T, DK, H, or K; xg is N, S, K, E, Y, D, I, or Q; xl 0 Is N, Y, S, D, G, or H; x 1 l is P; x 12 is R, I, or K; x 13 is T, I, M or A; yi is R, 1 Y2 is A, Y3 is F, Y4 is Y, is T, Y6 is T, Y7 is G, Y8 is R, Yg is I, is I, Yii is G, Y12 is D, F, P or W; S, or T; T, M, R, VH, or FT; A, R, N, V, I, K, E, E, 1, a, N, K, K, R, H, A, D, H, I, S, Y, or not present; M, or not present; D, Q, A, H, N, P, or not present; N, A, G, S, 1, or not preseit; or not present; R, L, S, E, Q, A, or not present; or not present; S, or not present; Y13 is I, M, ME, L, Or not present; Y14 is R, G, K, S, E, or not present; is 0, K, or R; Y16 is A; Y17 is H, Y, R, or Q; and wherein eithier Y4 is Y or Y6 is T.

8. The composition, according to claim 7, wherein X 1 is I; z is P; yi is R; and Y2 is A.

9. The composition, according to claim 7, wherein 921130,p:\oper~eAb44036-89.xsp,8O I*S~ 3 S.' '4 -I, 81 x 1 is I; x 3 is I; z is R; and Yl is R, The composition, according to claim 7, wherein z is P; Yl is R; Y2 is A; and Y3 is F.

11. A composition, according to claim 7, wherein xI is I; x 2 is H; x 3 is I; x 4 is R; x 5 is K; x 6 is R; x 7 is T; z is P; Yl is R; Y2 is A; Y3 is F; Y4 is Yp Y5 is T; Y6 is T; and Y7 is G. V i

12. The composition, according to claim 6, wherein said compound is circularized.

13. The composition, according to claim 6, wherein a and/or b comprise a peptide from an HIV principal neutralizing domain as hereinbefore defined.

14. The composition, according to claim 6, wherein said moiety capable of enhancing immunogenicity is a viral 921130,p:\oper\ejh,44036-89.rsp81 82 particle, microorganism, or immunogenic portion thereof. A prophylactic or therapeutic composition comprising a pharmaceutically acceptable carrier and/or diluent and immune globulin, monoclonal antibodies, and/or polyclonal antibodies generated by immunizing an appropriate animal such as a mouse, rat, horse, goat, human, or chimpanzee with a compound which comprises polypeptides which are not naturally occurring HIV envelope proteins, said polypeptides having the capability of eliciting, and/or binding with, neutralizing antibodies, said polypeptides comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant.

16. A prophylactic or therapeutic composition comprising antibodies generated by first immunizing an animal or human with at least one compound comprising a principal neutralizing domain as hereinbefore defined of an HIV variant followed by immunizing said animal or human with a mixture comprising compounds, where said compounds are not naturally occurring HIV envelope proteins, said compounds having the capability of eliciting, and/or binding with, neutralizing antibodies, said compounds comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant; or a compound which comprises polypeptides, where said polypeptides are not naturally occurring HIV envelope proteins, said polypeptides having the capability of eliciting, and/or binding with, neutralizing antibodies, said polypeptides comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant.

17. A prophylactic or therapeutic composition, comprising an antibody raised against a mixture comprising compounds, 930331,p:\opr\ej44036-89.rsp,82 83 where said compounds are not naturally occurring HIV envelope proteins, but having the capability of eliciting, and/or binding with, neutralizing antibodies, each of said compounds comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant.

18. A prophylactic or therapeutic composition, comprising a pharmaceutically acceptable carrier and/or diluent and an antibody raised against at least one compound having the capability of eliciting, and/or binding with, HIV neutralizing antibodies, said compound having the formula a x G z G y b wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; and z is P, A, S, Q, or L; a, b, x and/or y is from a peptide of the HIV principal neutralizing domain as hereinbefore defined; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant and wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T.

19. The composition, according to claim 18, wherein a and/or b comprises a peptide from an HIV principal neutralizing domain. An antibody raised against a compound, said compound having the formula a x G z G y b wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; 921130,p:\oper\e44036-89.1p,83 84 Z is P, A, S, 0, or L; and a, b, x and/or y is from a peptide of the HIV principal neutralizing domain as hereinbefore defined; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing intmunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a )eptide capable of stimulating T-cells, or a general immune stimulant.

21. The antibody, according to claim 20, wherein x is selected from x(Q) x is not present x(1) aX x(2) ax 2 xl x(3) ax 3 x 2 xl x(4) ax 4 x 3 x 2 xl ax 5 x 4 x 3 x 2 xl x(6) ax 6 x 5 x 4 x 3 x 2 xl x(7) x 7 x 6 x 5 x 4 x 3 x 2 xl X(8) ax 8 x 7 x 6 x 5 x 4 x 3 x 2 xl X(9) axgx 8 x 7 x 6 x 5 x 4 x 3 x 2 xl x~l) axl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 x 1 X(11) axllxl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl x( 12) axl 2 xllxl 0 x~x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl x( 13) ax 13 xl 2 ~xxgxx 8 x 7 x 6 x 5 x 4 x 3 x 2 xl y(Q) ay is not present y(l) al y(3) aY 3 y 2 Yl y(4) ayy 4 y 3 y 2 y ayy 5 y 4 y 3 y 2 y y(6) 6 y 5 y 4 y 3 y 2 yl y(8) aY 8 y 7 y 6 y 5 y 4 Y 3 y 2 yl 921l30,p:\operej 443-89.rsp,84 85 Y(9) ~Y9Y8Y7Y6YY4Y3Y2Y1 Y1OYY8Y7Y6Y5Y4Y3Y2Y1 Y(ll) yllY1OY9y8y76y5Y4Y3Y2Y 1 y(12) Y12YllY1OYgY7YyY5Yy43y2y1 y( 13) Y13Y12Y11Y1OYgY8Y7Y6Y5Y4Y3Y2Y1 y(14) Y14Y13Y12Yll1YlOY9yY 766yY4Y3Y2Y 1 Y1Y14Y13Y12Y11YoY9Y8yIy6yy4Y3Y2Y1 y(16) Y16Y15Y4Y13Y12Y 1Y1OY'9Y8Y7Y6Y5Y4Y3Y2Y 1 y(17) Y1Y16Y15Y14Y13Y12Y11Y1OyY8Y7Y6YSyg43y2y1 arid wherein x 1 is I, R, M, IQR, V, L, K, F, S, G, Y, SRG, or YQR; x 2 is H, R, Y, T, So P, F, N, A, K, G 4 or V; x 3 is I, L, M, T, V, E, G, F, or Y; X 4 is R, S, G, H, A, K, or not present; x 5 is K, R, I, N, Q, A, IR, RQ, or not present; x 6 is R, K, S, I, P, Q, E, G, or T; x 7 is T, K, V, I, A, R, P, or E; x 8 is N, NV, Y, KI, I, T, DK, H, or K; xg is N, S, K, E, Y, D, I, or Q; x 10 is N, Y, S, D, G, or H; x 11 is P; x 12 is R, I, or K; X 13 is T, I, M or A; Yj is R, K, 0, G, S, Y2 is A, V, N, R, K, Y3 is F, I, V, L, W, Y4 is Y, V, H, L, F, is T, A, V, 0, H, Y6 is T, R, I, Q, A, Y7 is G, E, K, R, T, Y8 is R, Q, E, K, D, yg is I, V, R, N, G, is I, T, V, K, M'l Yii is G, R, E, K, H Y12 is D, N, I, R, T .4I or T; T, S, F, P or W; Y, G, S, or T; S, 1, T, M, R, VH, or FT; I, S, Y, or not present; M, or not present; D, Q, A, H, N, P, or not present; N, A, G, 5, I, or not present; or not present; R, L, S, E, 0, A, or not present; or not present; or not preseni; Y13 is I, Y14 is R, ME, L, or not present; K, S, E, or not present; Il 92113,p operejA44036-89mp,85 86 is Q, K, or R; Y16 is A; Y17 is H, Y, R, or Q; and wherein either Y4 is Y or Y6 is T.

22. The antibody, according to claim 21, wherein said compound(s) are selected from the group consisting of a-Y-S-N-V-R-N-R-I-H-I-G-P-G-R-A-F-H-T-T-K-R-I -T-b; a-N-N-N-T-S-R-G-I-R-I-G-P-G-R-A--I-L-A-T-E-R-I -I-b; a-N-N-N-T-R-K-G-I-F-I-G-P-G-R-N-I-Y-T-T-G-N-I-I -b; a-N-T-R-K-S-I-R-I-Q-R-G-P-G-R-A-F-V-T-I-G-K-I-G-b; a-N-N-N-T-R-K-R-(I or or or or T)-G-Q-I-I-b; a-N-N-N-(I or or or or K) -V-I I-b; a-N-N-N-T-R-K-G-I-Y-V-G-S-G-R-(A or or H or or and a-T-R-Q-(R or or S)-L-Y7T-T-R-b.

23. A vaccine composition for generating a broadly neutralizing immunological response, said vaccine composition comprising a pharmaceutically aicceptable carrier and/or diluent and a mixture comprising compounds, where said compounds are not naturally occurring HIV envelope proteins, but have the capability of eliciting, and/or binding with neutralizing antibodies, said compounds comprising the principal neutralizing domain as hereinbefore dJefined, or a segment thereof, of HIV variants.

24. A vaccine composition, for generating a broadly neutralizing immunological response, said vaccine composition coomprising a pharmaceutically acceptable S*carrier an, i/or diluent, at hybrid comon.4 which comprises polypeptides, where said polypeptirdes are not naturally occurring HIV envelope proteins, but have the capability of eliciting, and/or binding with, neutralizing antibodies, 930331p:~oper\eAb44O3&89.rp,86 87 said polypeptides comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant. A vaccine composition comprising a pharmaceutically acceptable carrier and/or diluent and at least one compound having the capability of eliciting, and/or binding with, HIV neutralizing antibodies, said compound having the formula ax G z G y b wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; z is P, A, S, Q, or L; and a, b, x and/or y is from a peptide of the HIV principal neutralizing domain as hereinbefore defined; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiet." capable of enhancing immunogenicity, a peptide from an H.V principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant, and wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T. i 26. The vaccine composition, according to claim wherein said composition is formulated with an immunological adjuvant.

27. The vaccine, according to claim 25, wherein x is selected from x(0) x is not present x(1) x x(2) a x2x 1 x(3) x 3 x 2 x 1 x(4) a x 4 x 3 x 2 x 1 x 5 x 4 x 3 x 2 x 1 921130,p:\oper\ejk44036-89sp87 -88 x(6) x 6 x 5 x 4 x 3 x 2 xl x(7) ax 7 x 6 x 5 x 4 x 3 x 2 x 1 x(8) x 8 x 7 x 6 x 5 x 4 x 3 x 2 x 1 X(9) x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl Xc 10) xl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl X(11) xllxl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl x( 12) xl 2 xllxl 0 X 9 x 8 x 7 x 6 x 5 4 x 3 x 2 xl x( 13) xl 3 xl 2 Xllxl 0 X 9 X 8 X 7 x 6 X 5 x 4 x 3 x 2 Xl z is P, L, A, S, or Q; and y is selected from: y(0) -1 is not present Y(1 Y1 y(2) Y2Y1 y( 3 y3y2yl y5y4y3y2y1 y(6) y6y5y4y3y2yl y(7) y7y6y5y4y3y2y3. y(8) y8y7y6y5y4y3y2y1 Y(9) ygy8y7y6y5y4y3y2yl YlOy9y8y7y6y5y4y3y2yl Y(11) Ylly1Oy9y8y7y6y5y4y3y2y1 y( 12) yl2ylllOy9yBy7y6y5y4y3y2yl y( 13) Y13y12y11ylOygy8y7y6y5y4y3y2yI y( 14) Yl4yl3yl2y11ylOy9y8y7y6y5y4y3y2y1 y(6 Y15y4y3y2yllylQy9y8y7y6yy4y3y2y y(17) Y17yl6y15yl4yl3y12y11ylQy9yBy7y6y5y4y3y2y1 and wherein x, is I, R, M, IQR, V, L, K, F, S, G, Y, SRG, or YQR; x 2 is H, R, Y, T, S, P, F, N, A, K, G, or V; x3 is I, L, M, T, V, E, G, F, or Y; x 4 is R, S, G, H, A, K, or not present; xis K, R, I, N, 0, A, IR, RQ, or not present; x 6 is R, K, S, I, P, Q, E, G, or T; x7is T, K, V, I, A, R, P, or E 0 X 8 is N NV, Y, KI, I, T, DK, H, or K; x91s N, S, K, E, Y, D, 1, or Q; xl 0 is N, Y, S, D, G, or H; 921130,p:\oper\eAb44036-&9isp,88 89 x 11 is P; x1 2 is R, I, or K; x 1 3 is T, I, M or A; yi is R, K, Q, G, S, or T; Y2 is A, V, N, R, K, T, S, F, P or W; Y3 is F, I, V, L, W, Y, G, S, or T; Y4 is Y, V, H, L, F, S, 1, T, M, R, VH, or FT; is T, A, V, Q, H, I, S, Y, or not present; Y6 is T, R, I, Qf A, M, or not present; Y7 is G, E, K, R, T, D, 0, A, H, N, P, or not present; yB is R, Q, E, K, D3, N, A, G, S, I, or not present; y9 is I, V, R, N, G, or not present; yiO is I, T, V, K, M, R, L, S, E, Q, A, or not present; ylis G, R, E, K, H, or not present; Y12 is D, N, I, R, T, S, or not present; Y13 is I, M, ME, L, or not present; Y14 is R, G, K, S, E, or not present; is 0, K, or R; Y16 is A; Y17 is H, Y, R, or 0; and wherein either Y4 is Y or Y6 is T.

28. The vaccine, according to claim 27 wherein, X, is I; x2is H; *x 3 is I; K 4 isR; x 5 is K; x 6 is R;' x 7 is T; z is P; yj is R; Y2 is A; Y3 is F; Y4 is Y; is T; Y6 is T; and 921130,p:\oper\ejb,44036-S9.rspA 90 Y7 is G.

29. The vaccine, acoording to claim 27, wherein said compound(s) are selected from the group consisting of a-Y-S-N-V-R-N-R-I-H-I-G-P-G-R-A-F-H-T-T-K-R-I-T-b; a-N-N-N-T-S-R-G-I-R-I-G-P-G-R-A-I-L-A-T-E-R-I-I-b; a-N-N-N-T-R-K-G-I-F-I-G-P-G-R-N-I-Y-T-T-G-N-I- I-b; a-N-T-R-K-S-I-R-I-Q-R-G-P-G-R-A-F-V-T-I-G-K-I-G-b; a-N-N-N-T-R-K-R-(I or or W)-Y-(X or or T)-G-Q-I-I-b; a-N-N-N-(I or or or or K) a-N-N-N-T-R-K-G-I-Y-V-G-S-G-R-(A or or H or or and a-T-R-Q- (R or S (A or S -1-Y-T-Tr-R-b. The vaccine composition of claim 27 whareln sa.d compound(s) are capable of eliciting antibodies that bind to the sequence G-P-G-R-A-F.

31. The vaccine composition of claim 27 wherein said compound(s) are capable of eliciting antibodies that bind to the sequence I-G-P-G-R-A-F.

32. The vaccine composition of claim 27 wherein said compound(s) are capable of eliciting antibodies that bind to the sequence I-G-P-G-R-A.

33. The vaccine composition of claim 27 wherein said compound(s) are capable of eliciting antibodies that bind to the sequence I-a-I-G-P-G-R, wherein a is any of the amino acids.

34. The vaccine, according to claim 33, wherein a is H. The vaccine composition of claim 27 wherein said compound(s) are capable ol eliciting antibodies that bind IS' Q 921130,p:\oper~ejb44036-89.rsp,90 iT 0' 91 to the sequence I-a-I-G-P-G-R-A, wherein a is any of the amino acids.

36. The vaccine, according to claim 35, wherein a is H.

37. The vaccine composition of claim 27 wherein said compound(s) are capable of eliciting antibodies that bind to the sequence I-a-I-G-P-G-R-A-F, wherein a is any of the amino acids.

38. The vaccine, according to claim 37, wherein a is H.

39. The vaccine composition, according to claim wherein said compound(s) are circularized. The vaccine composition, according to claim wherein said compcund(s) comprise epitopes for more than one HIV variant.

41. The vaccine, according to claim 27, wherein x 1 is I; z is P; yl is R; and Y2 is A.

42. The composition, according to claim 27, wherein x 1 is I; x 3 is I; Sz is P; and Yl is R.

43. The composition, according to claim 27, wherein z is P; Yl is R; Y2 is A; and Y3 is F. S921130,p:oper 4403689 91 921 130,p:\oper\ejh,44036-89J.rp91 92

44. The vaccine, according to claim 27, wherein x is selected from the group consisting of x(7), x(10), x(ll); and y is selected from y(10), and y(ll). The composition, according to claim 27, wherein said compound has the following amino acid sequence: a-xl3-R-P-x 10 -Xg-x 8 -x-6- 7 -xxx 4 -x 3 -x 2 -x 1 G-z-G-Yl-y 2 -Y3-Y4- Y 5 -y 6 -y 7 -Y 8 -Y 9 -yl 0 -G-yl 2 -yl 3 -R-yl 5 -A-yl 7 -b.

46. A kit for use in detecting antibody against HIV in a biological fluid, said kit comprising: a compound, where said compound is not a naturally occurring HIV envelope protein, said compound having the capability of eliciting, and/or binding with neutralizing antibodies, said compound comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant; and a means for detecting complexes formed between said antibody and said compound, wherein said compound has the formula ax G z Gyb wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T.

47. The kit, according to claim 46, wherein x is selected from x(0) x is not present 921130,p:\oper\ejb,44036-89.,sp,92 93 x(1) X x(2) x 2 xl x(3) x 3 x 2 xl x(4) kc 4 x 3 x 2 xl x 5 x 4 x 3 x 2 xl x(6) x 6 x 5 x 4 x 3 x 2 xl x(7) x 7 x 6 x 5 N 4 x 3 x 2 xl X(8) Sx 8 x 7 x 6 x 5 x 4 x 3 x 2 x 1 X(9) x-ox 8 x 7 x 6 x 5 x 4 x 3 x 2 xl X(1O) xl 0 x 9 x 8 x 7 x 6 x 5 x 4 lc 3 x 2 xl X(11) S x 11 xl 0 x 9 x 8 x 7 x 6 x 5 x 4 x 3 x 2 xl x( 12) xl 2 xllxl 0 xgx 8 x 7 x 6 xc 5 x 4 x 3 x 2 xl x( 13) xl 3 xl 2 xllx 1 0 xcgx 8 x 7 x 6 x 5 x 4 x 3 x 2 xl z is P, L, A, S, or Q; and y is selected from: y(O) ay is not present Y(l) )r1 y(2) Yy y(3) Ey3y2yl y(4) y4y3Y2yl y5y4y3y2yl y(6) y6y5y4y3y2yi y(7) y7y6ySy4y3y2yl Y(S) y8y7y6y5y4y3y2yl Y(9) ygyBy7y6ySy4y3y2y1 Y1Oy9yBy7y6ySy4y3y2yl y(ll) YllylOygyBy7y6ySy4y3y2yl y( 12) Y12yl1y1OygyBy7y6ySy4y3y2y1 y(13) Yl3yl2yllylOygy8y7y6ySy4y3y2yl y( 14) Yl4yl3'Yl2ylly1Oygy8y7y6ySy4y3y2y1 t( 15) YlSyl4yl3Yl2yl1ylQy9y8y7y6y5y4y3y2vI y(1L6) Y16yl5Y14 Y13yi2y11yioYgy8y7y6ySy4y3y2yl y (17) Y17y1 6yl5li 4Y13Yl,2Yl lOy9y8yIy6ySy4y3Y2Y1 and wherein xl is I, R, M, IQR, V, 11j K, F, S, G, Y, SRG, or YQR; x 2 is H, R, Y, T, S, P, F, N, A, K, G, or V; x 3 is I, L, M, T, V, E, G, F, or Y; x 4 is R, S, G, H, A, K, or not present; 921 130,p:\oper\ejb44036-89.rsp,93 94 x 5 is K, R, N, Q, A, IR, RQ, or no~t present; x 6 is R, K, S, I, P, Q, E, G, or T; x 7 is T, K, V, 1, A, R, P, or E; x 8 is N, NV, KI, I, T, DK, H, or K; xgis N, S, K, E, Y, D, I, or Q; is N, Y, S, D, G, or H; x 11 is P; x12 is R, or K; x 1 3 is T, 1, M or A; yb is R, X, Q, G, S, or T; Y2 is A, V, N, R, K, T, S, F, P or W; Y3 is F, I, V, L, W, Y, G, S, or T; Y4 is Y, V, H, L, F, S, I, T, M, R, VH, or FT; is T, A, V, Q, H, I, S, Y, or not present; y6 is T, R, I, Q, A, M, or not present; Y7 is G, E, K, R, T, D, Q, A, H, N, P, or not present; y8 is R, Q, E, K, D, N, A, G, S, 1, or not present; yg is I, V, R, N, G, or not present; ylb is I, T, V, K, M, R, L, S, E, Q, A, or not present; yll is G, R, E, K, H, or not present; Y12 is D, N, I, R, T, S, or not present; Y13 i'1 1, M, ME, L, or not present; Y14 is R, G, K, S, E, or not present; is 0, K, or R, Y16 isA Y 1 7 is H, Y, R, or 0; and wherein either Y4 is Y or0 is T. *48. The kit, according to claim 46, wherein said comipound(s) are selected from the group consisting of a-YI-S-N-V-R-N-R-I-H-I-G-P-G-R-A-F-H-T-T-K-R-I-T-b; a-N-N-N-T-S-R-G-I-R-I-G-P-G-R-A- I-L-A-T-E-R-I-I-b; a-N-N-N-T-R-K-R- (I or, (Y :r W (X or or S(f) a-N-N-N-(I or or or or 921130,p:\oper~eh44036-r9).rsp,94 95 K)-V-I-Y-A-T-G-Q-I-I-b; a-N-N-N-T-R-K-G-I-Y-V-G-S-G-R-(A or or H or or and a-T-R-Q-(R or or S)-L-Y-T-T-R-b.

49. The immunological assay, according to claim 48, wherein said compound has the formula: a x G z G y b wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant. A method for generating broad neutralizing polyclonal or monoclonal antibodies, said method comprising immunization with a mixture comprising compounds, where said compounds are no* naturally occurring HIV envelope proteins, said compounds having the capability of eliciting, and/or binding with, neutralizing antibodies, where each -of said compounds comprises the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant.

51. A method for generating broad neutralizing polyclonal or monoclonal antibodies, said method comprising immunization with a compound which comprises polypeptideo having the capability of eliciting, and/or binding with, neutralizing antibodies, where each of said polypeptides comprises the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant. tr 331,\ 93l,p:\oprejb,44Q36-89.z*95 95 I-I-b; a-N-N-N-T-R-K-G-I-Y-V-G-S-G-R-(A or or H or or and a-T-R-Q-(R or or S)-L-Y-T-T-R-b. 49. The immunological assay, according to claim 48, wherein said compound has the formula: axG z Gyb wherein x is 0 to 13 amino acids in length; y is 0 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant. A method for generating broad neutralizing polyclonal or monoclonal antibodies, said method comprising immunization with a mixture comprising compounds, where said compounds are not naturally occurring HIV envelope proteins, said compounds having the capability of eliciting, and/or binding with, neutralizing antibodies, where each of said compounds comprises the principal 4; neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant. 51. A method for generating broad neutralizing polyclonal or monoclonal antibodies, said method comprising immunization with a hybrid compound which comprises polypeptides having the capability of eliciting, and/or binding with, neutrali7ing antibodies, where each of said polypeptides comprises the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant. 921130,p:\oper\eh,44036-89jsp,95 96

52. A method, according to claim 51, said method comprising immunization with at least one compound having the capability of eliciting, and/or binding with, neutralizing antibodies, said compound having the formula a x G z G y b wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, bu't not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant and where either the fourth amino acid of said y is Y or the sixth amino acid of said y is T.

53. A method of detecting antibody against HIV in a biological fluid, comprising the steps of: incubating a compound, other than a naturally occurring HIV envelope protein, said compound having the capability of eliciting, and/or binding with, neutralizing t antibodies, said compound comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant; detecting complexes formed between said antibody and said compound wherein said compound has the formula: axGzGyb wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune S921130,poper\ 3689p96 97 stimulant, and wherein either the fourth amino acid of sad y is Y or the sixth amino acid of said y is T.

54. The method, for stimulating a lymphocyte prolif2 r tive response in humans which comprises treating humans in need of stimulation of a lymphocyte proliferative response ith at least one compound, said compound is not a naturally occurring HIV envelope protein, said compound having the capability of eliciting, and/or binding with, neutralizing antibodies, said compound comprising the principal neutralizing domain, or a segment thereof, of an HIV variant, wherein said compound has the formula a x G z G y b wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T.

55. The method for treatment of HIV infection, said method comprising administering to an animal or human in need of such treatment a composition comprising one or more compounds wherein said compounds are not naturally occurring HIV envelope proteins, but have the capability of eliciting, and/or binding with, neutralizing antibodies, each of said compounds comprising th. principal neutralizing domain, as hereinbefore defined, or a segment thereof, of an HIV variant, wherein said compounds have the formula axGz Gyb wherein x is 0 to 13 amino acids in length; S' 921130,p:\oper\ejb44036-89.rsp,97 sTOA£ 98 y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant and wherein either the fcurth amino acid of said y is Y or the sixth amino acid of said y is T.

56. A method for assaying a biological fluid for the presence of an HIV variant-specific protein, said method comprising contacting said fluid with an antibody specific for compounds wherein said compounds are not naturally occurring HIV envelope proteins, but have the capability of eliciting, and/or binding with, neutralizing antibodies, said compounds comprising the principal neutralizing domain, as hereinbefore defined, or a segment thereof, of an HIV variant; and detecting immune complexes as a measure of said variant in said fluid, wherein said compounds of part (c) Shave the formula a x G z G y b wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant and wherein either the fourth amino acid of said y is Y or the sixth amino acid of said y is T. 921130,p:\oper\ejh,44036-89.rsp,98 99

57. A method of in vitro lymphocyte stimulation comprising treating lymphoid cells from immune animals with at least one compound, other than naturally occurring HIV envelope proteins, said compound having the capability of eliciting, and/or binding with, neutralizing antibodies, said compound comprising the principal neutralizing domain as hereinbefore defined, or a segment thereof, of an HIV variant.

58. The method, according to claim 57, where said compound has the formula a x G z G y b wherein x is 0 to 13 amino acids in length; y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain as hereinbefore defined, a peptide capable of stimulating T-cells, or a general immune stimulant.

59. A method of identifying polyclonal or monoclonal antibodies which are useful in prophylaxis or therapy, said method comprising screening antibody-producing cells for ability to bind with at least one compound, other than naturally occurring HIV envelope proteins, said compound having the capability of eliciting, and/or binding with, neutralizing antibodies, said compound comprising the principal neutralizing domain as hreinbefore defined, or a segment thereof, of an HIV variant. The method, according to claim 59, wherein said compound has the formula a x G z G y b wherein x is 0 to 13 amino acids in length; 921130,p:\oper\e4403689rsp,99 100 y is 6 to 17 amino acids in length; and z is P, A, S, Q, or L; and either a or b, but not both, may be omitted; either a or b individually may comprise any one of the following: cysteine, a protein or other moiety capable of enhancing immunogenicity, a peptide from an HIV principal neutralizing domain, a peptide capable of stimulating T- cells, or a general immune stimulant.

61. A synthetic gene which encodes a polypeptide wherein said polypeptide comprises a principal neutralizing domain as hereinbefore defined of more an one HIV variant.

62. A synthetic gene which codes a polypeptide wherein said polypeptide comprises neutralizing epitopes from a principal neutralising domain as hereinbefore defined of more than one HIV isolate.

63. The synthetic gene, according to claim 62, wherein said polypeptide comprises neutralizing epitopes from 2 to HIV isolates.

64. The synthetic gene, according to claim 62, wherein one of said neutralizing epitopes is from the HIV isolate designated HIV-MN. The synthetic gene, according to claim 62, wherein said polypeptide comprises neutralizing epitopes from the HIV isolates which have been designated HIV-MN, HIV-IIIB, I. HIV-RF, HIV-SC, and HIV-WMJ1.

66. The synthetic gene, according to claim 62, wherein the regions encoding neutralizing epitopes from different HIV isolates are separated by amino acid spacers.

67. The synthetic gene, according to claim 66, wherein said amino acid spacers are glycines. 7U 930625,p:\oper\ejh,4436-89.p,100 U93OX25,p:\oper\ejh,44036-89sp,100 101

68. The synthetic gene, according to claim 62, wherein said gene codes for a polypeptide having the amino acid sequence shown in Table 13, or an equivalent amino acid sequence.

69. The synthetic gene, according to claim 62, wherein said gene codes for a fusion polypeptide. The synthetic gene, according to claim 69, wherein said gene codes for a polypeptide having the amino acid sequence shown in Table 13A, or an equivalent amino acid sequence.

71. A compound comprising neutralizing epitopes from a neutralizing domain as hereinbefore defined of more than one HIV isolate, said compound comprising a fusion protein.

72. The compound, according to claim 71, wherein said compound comprises neutralizing epitopes from 2 to 20 HIV isolates.

73. The compound, according to claim 71, wherein one of t .said neutralizing epitopes is from the HIV isolate designated HIV-MN. itt

74. The compound, according to claim 71, wherein said compound comprises neutralizing epitopes from the HIV i i isolates which have been designated HIV-MN, HIV-IIIB, HIV- RF, HIV-SC, and HIV-WMJ1.

75. The compound, according to claim 71, wherein said neutralizing epitopes are separated by amino acid spacers.

76. The compound, according to claim 75, wherein said amino acid spacers are glycines. 930625,p:\oper\ej4436-89sp,101 102

77. The compound, according to claim 71, wherein said compound has the amino acid sequence shown in Table 13, or an equivalent amino acid sequence.

78. The compound, according to claim 71, wherein said compound is a fusion polypeptide.

79. The compound, according to claim 78, wherein said compound has the amino acid sequence shown in Table 13A, or an equivalent amino acid sequence. A prophylactic or therapeutic composition, comprising immune globulin, monoclonal antibodies, and/or polyclonal antibodies generated by immunizing an appropriate animal with a composition comprising a multi-epitope compound wherein said multi-epitope compound comprises neutralizing epitopes from a principal neutralizing domain as hereinbefore defined of from 2 to 20 HIV isolates, wherein one of said isolates is HIV-MN.

81. The composition, according to claim 80, wherein said multi-epitope compound comprises neutralizing epitopes from the HIV isolates designated HIV-MN, HIV-IIIB, HIV-RF, HIV- SC, and HIV-WMJ1.

82. A method for generating broad neutralizing polyclonal or monoclonal antibodies, said method comprising immunizing an appropriate animal with a composition comprising a multi-epitope compound wherein said multi-epitope compound comprises neutralizing epitopes from a principal neutralizing domain as hereinbefore defined of from 2 to HIV isolates, wherein one of said isolates is HIV-MN. 930625,p:\oper\ ej,4436-89p, 102 103

83. The method, according to claim 82, wherein said multi- epitope compound comprises neutralizing epitopes from the HIV isolates which have been designated HIV-MN, HIV-IIIB, HIV-RF, HIV-SC and HIV-WMJ1.

84. A method for prophylaxis of therapy of HIV infection, said method comprising administering to an animal or human in need of such prophylaxis or therapy a pharmaceutical composition comprising immune globulin, monoclonal antibodies, and/or polyclonal antibodies generated by immunizing an appropriate animal with a composition comprising a multi-epitope compound wherein said compound comprises neutralizing epitopes from a principal neutralizing domain as hereinbefore defined of from 2 to HIV isolates, wherein one of said isolates is HIV-MN. The method, according to claim 84, wherein said multi- epitope compound comprises neutralizing domains from the HIV isolates which have been designated HIV-MN, HIV-IIIB, HIV-RF, HIV-SC and HIV-WMJ1.

86. A process for stimulating a lymphocyte proliferative response in humans which comprises treating humans in need of stimulation of a lymphocyte proliferative response with a composition comprising a multi-epitope compound wherein said compound compri.es a neutralizang domain from a principal neutralizing domain as hereinbefore defined of from 2 to 20 HIV isolates, wherein one of said isolates is HIV-MN.

87. The process, according to claim 86, wherein said multi-epitope compound comprises neutralizing epitopes from the HIV isolates which have been designated HIV-MN, HIV- IIIB, HIV-RF, HIV-SC, and HIV-WMJ1. 930625,p:\oper\jh,44036-89.rsp,103 104

88. A compound comprising neutralizing epitopes from a principal neutralizing domain as hereinbefore defined of from more than one HIM isolate, said epitopes being linked by a covalent non-peptide bond. DATED this 25th day of June, 1993 REPLIGEN C4RORATION by its rratent JAttorneys DAVIES dOLLIS0N CAVE 930625,p:\oper\ji,440A3689.rsp,1O4